supporting information - pnas...2014/02/26 · at a concentration of 20 μg/ml, and anti-ck8,...

Supporting InformationSpring et al. 10.1073/pnas.1319493111SI TextFluorescence Microendoscope. The custom-built microendoscope(1) uses a blue light-emitting diode (LXHL-LR5C; Luxeon StarLEDs) excitation source, a 10× objective (NT46-144; EdmundOptics), and a dichroic mirror (500dcxr; Chroma Technology).The beam was focused onto the proximal end and transmitted tothe distal end of a 1.5-m-long, 800-μm-diameter, and 0.35-N.A.flexible coherent fiber bundle (IGN-08/30; Sumitomo Electric),delivering an optical power of 0.25 mW during contact modeimaging of the sample. The coherent fiber bundle consists of30,000 cores. The core-to-core spacing is ∼4.4 μm, sufficient forcellular resolution imaging (1). Fluorescence emission was col-lected by the fiber probe through an emission filter (D700/40;Chroma Technology) onto an electron-multiplying CCD camera(Cascade 512B EMCCD; Photometrics), which has a quantumefficiency of ∼90% at the spectral region of the benzoporphyrinderivative (BPD) fluorescence emission peak (690–700 nm). TheEM gain and exposure time were fixed at 3,000 V and 100 msthroughout the experiments. Analysis of USAF test target im-ages determined 1.6-μm x–y sampling.

In Vitro Validations of Cet-BPD Specificity and ImmunofluorescenceStains. The specificity of Cet-BPD(1:7), anti-human cytokeratin 8(CK8) (clone LP3K; R&D Systems), anti-mouse CD45 (clone30-F11; R&D Systems), anti-human and -mouse epidermalgrowth factor receptor (EGFR) (Cell Signaling Technology;D38B1) monoclonal antibodies (mAbs) were tested in T47D(human epithelial ductal carcinoma; low EGFR expression),A431 (human epidermoid carcinoma; abnormally high EGFRexpression), OVCAR5 [epithelial ovarian cancer (EOC); mod-erate EGFR expression relative to A431—see also Fig. 4E andSI Text, Western Blots below], CT26.WT (mouse colorectal car-cinoma; moderate EGFR expression), and J774.2 (mousemonocyte macrophage) cell lines (Fig. S4). All cell lines wereplated in multiwell plates with coverslip bottoms (Greiner Bio-One) and allowed to grow overnight. The mAbs were conjugatedusing mAb-labeling kits (Life Technologies): anti-CK8, anti-protein tyrosine phosphatase, receptor type, C (CD45), and anti-EGFR mAb were conjugated to the Alexa Fluor (AF) dyesAF647, AF488, and AF568, respectively. Cet-BPD was preparedat a concentration of 20 μg/mL, and anti-CK8, -EGFR, and-CD45 stains each at 10 μg/mL. All mAbs were diluted in buffer(Dako Antibody Diluent with Background Reducing Compo-nents), and cells were incubated with blocking buffer (DakoProtein Block) before staining. Cet-BPD, anti-CD45, and anti-EGFR mAbs were incubated with living cells, whereas anti-CK8mAb was incubated with fixed cells. Cells were incubated withthe immunostains for 2 h at 37 °C and mounted using SlowFadeGold Antifade Reagent (Life Technologies). Imaging was per-formed with an Olympus FV1000 confocal microscope witha 40× objective. Excitation of BPD, AF488, AF568, and AF647was carried out using 405-, 488-, 559-, and 635-nm lasers, re-spectively. Lasers were scanned sequentially to reduce channelcrosstalk. The laser, photomultiplier tube detector, and pinholesettings, as well as brightness-contrast adjustment settings fordisplay, were kept constant for each fluorophore when imagingdifferent cell lines. For competition experiments, cells were in-cubated with either 2 mg/mL unconjugated cetuximab (Bristol-Myers Squibb) or trastuzumab (Genentech) for 5 h at 37 °Cbefore staining with Cet-BPD. Cet-BPD was diluted to 20 μg/mLand incubated with cells for 2 h before imaging.

Hyperspectral Imaging of the Peritoneal Cavity. Hyperspectralfluorescence images of the peritoneal cavity were acquired usinga CRi Maestro system. A 465-nm bandpass excitation filter,a 515-nm long-pass emission filter, and a liquid crystal tunableemission filter were used to acquire hyperspectral fluorescenceimages from 520 to 850 nm in 10-nm intervals. A custom, batch-processing Matlab routine performed pixel-by-pixel linearspectral deconvolution. Basis shape spectra for spectral unmixingwere acquired as follows. (i) Autofluorescence basis spectra(including the bowel, stomach, bladder, stomach, and skin) weremeasured before immunoconjugate injection. (ii) BPD basisspectra were measured from BPD dissolved in PBS with orwithout 2% Triton X-100, a detergent that enhances BPD sol-ubility. To verify accurate spectral unmixing, the reduced χ2 wascalculated for the spectral fit at each pixel based on the noisestatistics (a measured variance-signal curve) of the CCD cameraand the fit residuals. All images were background corrected (tosubtract dark current) and normalized by their exposure timefor global comparison. Verteporfin and Cet-BPD were admin-istered at equivalent BPD doses (2 mg·kg−1 body weight). Theimages in Fig. 2C were acquired 2 h post i.p. administration ofverteporfin or 48 h for the immunoconjugate Cet-BPD(1:7) onday 23 following tumor inoculation.

BPD Quantification by Tissue Extraction. As a gold standard,resected tissues were pulverized and dissolved in Solvable (Per-kin-Elmer) to measure the BPD fluorescence signal per gramtissue using a fluorometer, as detailed previously (2). This pro-cedure eliminates tissue optical properties and, therefore, servesas a validation of in vivo BPD quantification. The extractionexperiments were performed on EOC mice 10 d following tumorinoculation, and the tissue sites were categorized as either tumor(pelvic omentum, uterus, subgastric omentum, bowel mesentery,and diaphragm), or control no-tumor (spleen, liver, kidney, andbowel) based on the known dissemination pattern of the ortho-topic EOC mouse model (3). All data were background sub-tracted on a tissue site-specific basis, using extraction data frommice not injected with BPD. To report BPD fluorescence pergram tissue and tumor-to-tissue values (Fig. 2 D and E), datafrom 8, 12, and 24 h for Cet-BPD and for 2 and 4 h for verte-porfin were binned and averaged to reduce noise. Further noisesuppression is possible by fitting the temporal dynamics toa pharmacokinetic model (SI Text), which indicates higher tu-mor-to-tissue ratios for Cet-BPD as discussed in the text. Ver-teporfin and Cet-BPD were administered at equivalent BPDdoses (2 mg·kg−1 body weight).

Pharmacokinetic Model. Pharmacokinetic data for verteporfin andCet-BPD—measured by fluorescence microendoscopy and tissueextraction—were fit to a simple pharmacokinetic model a·(e−k·t −e−j·t), where k and j are the elimination and absorption rateconstants, a is a coefficient dependent on the administered BPDdose as well as its bioavailability, and t is time postinjection.

Confocal Imaging of Freshly Excised Tissues. Following i.p. injectionof immunoconjugate, freshly excised peritoneal wall specimenswere immediately blocked, followed by immunofluorescencestaining (10–16 μg/mL for each antibody), and rinsed with freshPBS. The tissue was then mounted on a coverslip bottom dish(Matek). Confocal fluorescence imaging was performed using anOlympus FluoView 1000 confocal microscope with 10×, 40×(water immersion), and 60× (water immersion) objectives. Anti-

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mouse CD45 mAb-AF488, anti-mouse PECAM-1 (CD31; clone390; Millipore) mAb-AF568, and anti-human CK8 mAb-AF647were used to image immune cells, endothelial cells (ECs), andEOC cells, respectively. Confocal imaging and staining wereperformed as described above. The lateral and axial resolutionswere 0.2 and 2 μm, respectively. The stage was stepped in 1- to2-μm increments to acquire z stacks for determining colocalizationcoefficients. BPD and EOC objects less than 30 μm in dimensionwere excluded from the receiver operating characteristic (ROC)analysis. Analyses were performed using a custom, batch-processingMatlab (Mathworks) routine. The tumor classification and 3D co-localization tests used objective intensity thresholds set to reject99.5% of the background signal in each of the EOC, EC, andimmune cell channels (Fig. S3 B and C). The statistical significanceof the 3D colocalization computation was confirmed by scramblingimages of the Cet-BPD channel (Fig. S7).

Western Blots. Harvested monolayer cells were incubated withradioimmunoprecipitation assay lysis buffer (Thermo Scientific)containing phosphatase and protease inhibitors (Sigma-Aldrich).Clear supernatant protein lysates were preserved and quantifiedusing a Pierce BCA Protein Assay kit (Thermo Scientific). Thefollowing primary antibodies were used: rabbit anti-EGFR mAb(clone 15F8; Cell Signaling Technology), rabbit anti-HER2/ErbB2antibody (no. 2242; Cell Signaling Technology), and rabbit anti-β-actin mAb (clone 13E5; Cell Signaling Technology). The sec-ondary antibody was anti-rabbit IgG horseradish peroxidase-conjugated antibody (7074; Cell Signaling Technology). Horse-radish peroxide signal from protein bands was detected using theImmuno-Star AP Chemiluminescence Kit (Bio-Rad). Horserad-ish peroxide signals from proteins of interest were normalized totheir respective β-actin loading controls for densitometry. EGFR-negative (T47D) and -positive (A431; abnormally high EGFRexpression as shown in Fig. 4E, Upper, labeled “EGFR+

”)monolayer cell lines were used to validate the anti-EGFR pri-mary and secondary antibodies. Due to high levels of EGFRexpression in A431 cells, less protein was loaded. Similarly,HER2-negative (MCF7) and -positive (SKBR3) cell lines wereused to validate anti-HER2 antibodies.

Quantitative RT-PCR Measurement of Micrometastatic Burden.Quantitative reverse transcription–PCR (qRT-PCR) was vali-dated (Fig. S9A) and used to measure the total number of humanEOC cells within the peritoneal cavity from the level of human andmouse glyceraldehyde 3-phosphate dehydrogenase (GAPDH)housekeeping genes. This approach was inspired by a previousreport (4). However, here, we used reverse transcription to targetRNA rather than DNA, which is specific to viable human cancercells (RNA is rapidly metabolized upon cell death). Entire peri-toneal cavities were collected at the treatment endpoints and snapfrozen in liquid nitrogen. Frozen peritoneal cavities were pulver-ized and homogenized, followed by RNA extraction (RNAeasyPlus Mini Kit; Qiagen). Human and mouse GAPDH gene weremeasured using custom synthesized primers (Invitrogen). For eachspecimen, the cycle threshold (Ct) from human GAPDH gene wasnormalized by Ct from mouse GAPDH gene. The normalized Ctwas quantified into number of cancer cells using a standard curvegenerated with a set of peritoneal cavity lysates from no-tumorcontrol mice mixed with different numbers of human EOC cells.

Note S1: Fiber Optic Light Delivery to Tumors Deep Within the Body.An off-cited misconception of photoactivated therapies is thatthey have limited application due to the finite tissue penetrationdepth of light. However, miniature fiber optic light conduits makeit possible to reach tumors deep within the human body (e.g., asillustrated in Fig. 1F) such that photodynamic therapy of me-tastases and deep tumors is practical with modern technology.This is possible because light transport is made efficient using

diffusing tip fibers and scattering media (intralipid emulsion) tospread the light over large areas, such as the entire pleural andperitoneal cavities. As an example, our prior preclinical studies(with “always-on” photodynamic agents) demonstrated photo-activated tumor destruction explicitly in hepatic, pelvic, subgastric,diaphragmatic, spleen, and bowel sites (5). In fact, photodynamictherapy is clinically approved for treatment of bladder, lung, andesophageal cancers (6, 7). Furthermore, clinical trials have dem-onstrated feasibility, safety, and efficacy for photodynamic treat-ment of primary tumors in the pancreas (8, 9), locally malignantglioblastoma multiforme in the brain (10), and disseminated,metastatic tumor deposits spread throughout the pleural (resultingfrom non–small-cell lung cancer) (11) and peritoneal (resultingfrom ovarian cancer as well as malignancies of the gastrointestinaltract) cavities (12). The enhanced tumor selectivity achieved bytumor-targeted, activatable photoimmunotherapy (taPIT) offerspromise to push this forward by enabling safe use of intense, diffuselaser light for targeted, wide-field treatment of micrometastasesembedded in vital tissues. At the moment, the drug and light dosesare restricted by nonspecific toxicities when using diffuse irradia-tion to efficiently treat disseminated disease. However, the fact isthat light delivery is feasible for photodynamic treatment of cancerdeposits in the following anatomical sites: the esophagus (6, 7),bladder (6, 7), brain (10), bone (13), lungs (6, 7, 14), pancreas (8,9), and those studding the peritoneal organs (12). Here, we dem-onstrate these concepts in a sophisticated mouse model of cancermicrometastases characterized by disseminated micronodular tu-mor deposits studding vital organs within the peritoneal cavity. Thecytotoxic component of our activatable immunoconjugate is trig-gered by near-infrared light, which has sufficient tissue penetrationto treat microscopic tumors.

Note S2: Rationale for the Use of Cetuximab and the Potential to UseOther Tumor-Targeting mAbs for taPIT and Micrometastasis Imaging.Cetuximab is in clinical use for the treatment of colorectal canceras well as head and neck cancer. It is a chimeric (mouse/human)mAb directed against EGFR (ErbB1/HER1). EGFR is a receptortyrosine kinase (RTK) of the ErbB family that is overexpressedand abnormally activated in a number of epithelial tumors (15),including EOC (16). EGFR activation leads to downstream in-tracellular signaling that promotes cell proliferation, angiogen-esis, and other survival and tumor growth signaling pathways(15). Cetuximab therapy has been shown to promote cell cyclearrest and apoptosis while inhibiting angiogenesis (15). Cetux-imab is an IgG1 isotype also capable of mediating antibody-de-pendent cellular toxicity (15). A limitation of cetuximab, anda side effect of all anti-EGFR treatments, is the development ofa dose-dependent skin rash within the first few weeks of treat-ment (17). The rash is reversible and generally mild enough tocontinue therapy, but some patients (8–17%) do have more se-vere reactions that necessitate dose reduction or interruption(17). The mice used in this study did not develop rashes or anyovert signs of skin phototoxicity (no attempts were made to avoidnormal room light exposure).As proof of concept, we primarily synthesized cetuximab-based

immunoconjugates stemming from our observation of synergistictumor reduction when combining cetuximab with photodynamictherapy using verteporfin (18). However, the concepts of taPITand micrometastasis imaging reported here can be applied toa variety of tumor biomarkers and to address tumor heteroge-neity in biomarker expression because many other tumor-tar-geted antibodies and antibody fragments are available (19). Thisis important because it is unlikely that a single antigen target ortherapy will be sufficient to clear advanced disease. In fact, thecurrent trend is to use mixtures of chemotherapy for ovariancancer and many other cancers. Other mAbs for taPIT includepanitumumab, a fully humanized mAb of the IgG2 isotype, di-rected against EGFR and in the clinic. Panitumumab lacks the



cytotoxic component of cetuximab but shares many other mech-anisms of action with cetuximab, and it has been applied for PIT(20). As another example, trastuzumab, also applied here, can beused to target HER2 (ErbB2)-overexpressing cancer cells. Inaddition to the ErbB family of receptors, there exists mAbsin clinical trials for a number of other cancer cell surface bio-markers including hepatocyte growth factor/mesenchymal-epi-thelial transition factor RTK, epithelial cell adhesion molecule,and folate receptor α. mAb fragments (e.g., ranibizumab) andsmall peptides are also emerging and have the advantage ofbeing considerably smaller than full mAbs (for deeper tissuepenetration) but lack the cytotoxic component of IgG1s (withother pharmacokinetic and pharmacodynamic alterations rela-tive to full mAbs).

Note S3: Rationale for the Use of Verteporfin to Facilitate taPIT ofDisseminated Metastases.Verteporfin (BPD) is a photosensitizingagent for photodynamic therapy clinically approved for thetreatment of the wet form of age-relatedmacular degeneration (7,21). Broadly, photodynamic therapy with unconjugated photo-sensitizing agents, including verteporfin, is being applied forcancer patients for whom all other options have failed. Light canbe delivered intraoperatively or endoscopically and results insignificant improvements (7, 9, 12, 22). Verteporfin is currentlyundergoing clinical trials for the treatment of pancreatic ductaladenocarcinoma. A recent phase I clinical trial established safetyand a 100% response rate with a 1- to 2-cm zone of necrosis forsingle-fiber interstitial photodynamic therapy of locally advancedpancreatic tumors (8). This is of particular promise becausepancreatic ductal adenocarcinoma is a deadly disease (23) andnotoriously resistant to chemotherapy (24) such that currenttreatments are only palliative (25). However, nonspecific uptakeof photodynamic agents in the bowel and bowel toxicity havebeen a major obstacle to the clinical translation of photodynamictherapy for treating metastatic disease in the peritoneal cavity(26), a key feature of ovarian cancer and a common problemamong gastrointestinal malignancies. Poor tumor-to-bowel ratiosof drug uptake were identified in these clinical trials of i.p.photodynamic therapy using always-on photodynamic agents(26). Our prior phototoxicology studies also identified boweltoxicity as problematic for i.p. photodynamic therapy with ver-teporfin (always-on BPD) in the mouse model of metastaticovarian cancer (5). The tumor selectivity of taPIT overcomes thisbowel toxicity.

Note S4: Comments on the Importance of EGFR as a TherapeuticTarget and as a Prognostic Indicator. The importance of EGFRas a therapeutic target has been critically evaluated in recentyears. First, EGFR is a viable target for taPIT because it isfrequently overexpressed by human ovarian cancers (16, 27).Second, it is well known that EGFR monotherapy has limitedefficacy due to a myriad of escape mechanisms (28) in similarityto the frequent escape of cancer cells from chemotherapy and allother therapies. This is not reason to give up. To address thisproblem, rationally designed combination therapies are neededto harness EGFR-targeted agents. This is exactly the point of thepresent study. That is, taPIT does not rely on EGFR mono-therapy, but rather uses the photodynamic sensitization of cancercells to anti-EGFR agents (18, 29, 30) while providing a powerfulphotocytotoxic arm that appears to be effective against re-current, drug-resistant disease (31). Photodynamic therapy andPIT are finite treatment modalities that lead to a burst in celldeath and survival signaling (e.g., EGFR activation and secretionof vascular endothelial growth factor) (32, 33), which primes thecancer cells for concomitant molecular-targeted therapy (e.g.,cetuximab) and leads to synergistic reductions in the tumor (7,18, 34). Third, an elegant clinical report by Psyrri et al. (16)developed improved immunostaining methods and rigorously

calibrated immunohistochemistry techniques for quantifyingEGFR levels. This definitive study found that EGFR expressionis a statistically significant adverse prognostic indicator for hu-man ovarian cancers—increased EGFR expression level asso-ciates with decreased survival (16). The increase in mediansurvival is more than 2 y for low EGFR expressing ovariancancers (>34 mo compared with 12 mo for high–EGFR-expressingovarian cancers; hazard ratio = 8.86, P = 0.0001) (16). Thehazard ratio for EGFR expression in ovarian cancer is moremodest according to the metaanalysis of de Graeff et al. (35);however, this metaanalysis includes many potentially flawedimmunohistochemistry studies of EGFR expression (11 of 15studies rely on immunohistochemistry). Psyrri et al. (16) attrib-uted prior conflicting results in the literature to poor stainingand qualitative analysis methods.

Note S5: Tumor-Targeted Activation of Both BPD Fluorescence andPhototoxicity. Both BPD fluorescence (photon emission from thesinglet state) and photocytotoxicity (energy transfer from the tripletstate to create singlet oxygen and other cytotoxic species) are ac-tivated upon lysosomal proteolysis of the internalized immuno-conjugate. Fluorescence and photocytotoxicity quenching bothresult upon loading BPD onto the antibody due to BPD stackinginteractions when these molecules are held in close proximity (2).This phenomenon is commonly termed “ground state quenching,”“contact quenching,” or “static concentration quenching” in theliterature (36). Upon cellular binding and lysosomal proteolysis ofthe immunoconjugate, BPD fluorescence and phototoxicity arerestored (2). Quenching is released upon antibody degradationand the subsequent physical separation of the BPD molecules,diminishing their propensity to stack and self-quench.On a theoretical basis, quenching of the singlet-excited state is

expected to also quench triplet-excited state photochemistry be-cause the triplet-excited state generally arises from the singlet statevia intersystem crossing (36). Direct excitation of the triplet-excitedstate (from the singlet ground state) is a spin-forbidden transitionof very low probability and is generally negligible, and the rate ofintersystem crossing is generally stable, although spin-orbit cou-pling can be increased in the presence of heavy atoms (36), and,for some chromophores, alterations in the rate of intersystemcrossing have been observed and attributed to microenvironment-induced shifts in the relative singlet-triplet energy separation.To illustrate these concepts mathematically, the excited state

lifetime (or the fluorescence lifetime, τ) is inversely proportionalto the sum over all rates of deexcitation:

τ=1

Piki;

whereP

i ki is the sum over all rates of the individual deexcita-tion pathways including fluorescence (kf ), internal conversion(kic), and intersystem crossing (kisc). Similar expressions can bederived for the fluorescence and triplet quantum yields. Thefluorescence quantum yield, Φf , is defined as follows:

Φf =kfPiki;

where kf is the rate of deexcitation via fluorescence emission.The triplet quantum yield, ΦT , is defined as follows:

ΦT =kiscPiki;

where kisc is the rate of intersystem crossing from the singlet tothe triplet state. Upon antibody conjugation, a new quenching



mechanism is introduced such that the fluorescence and tripletquantum yields become the following:

Φf =kf

kscq +P

i≠scqki; and ΦT =

kisckscq +

Pi≠scqki

:

Here, kscq is the rate constant for static concentration quenchingintroduced upon antibody conjugation and

Pi≠scq ki is the sum

over all other rate constants. The rate constant for static concen-tration quenching dominates the denominator. Thus, both thefluorescence and triplet quantum yields are quenched until pro-teolysis of the immunoconjugate; i.e., when kscq goes to zero, thefluorescence and triplet yields increase. An excellent reference forthe full derivation of these relationships from first principles is thetextbook by Valeur and Berberan-Santos (36).Regarding empirical evidence, our prior works (characterizing

the activatable immunoconjugates in vitro) show that both thesinglet state fluorescence and triplet state phototoxicity arequenched until the cells have time to internalize and process theimmunoconjugates, releasing the BPD (2, 37). That is, cell killing(via light irradiation) is not observed until 6–40 h after incubationwith the immunoconjugates, which corresponds to the time frameobserved for immunoconjugate binding, internalization, and deg-radation via lysosomal proteolysis (immunoconjugate degradationwas measured by gel electrophoresis of cell lysates) (37). Further-more, fluorescence quantum yield and fluorescence lifetime meas-urements, which report singlet state quenching, demonstrated thatthe singlet-excited state of BPD is quenched upon antibodyconjugation and the quenching efficiency increases with in-creased BPD loading onto the antibody (i.e., kscq increases withincreased BPD loading) (2). Finally, a study characterizingphotophysical properties of BPD found that self-quenchingupon aggregation in an aqueous suspension (without serumproteins or lipid) induces a strong reduction in both the fluo-rescence (∼26-fold) and the triplet state quantum yields (>24-fold; the triplet state is not detectable upon aggregation) (38).

Note S6: Residual Tumor-Associated Inflammatory Cells PosttreatmentUptake Cet-BPD. The lack of perfect cancer cell recognition in vivois due to Cet-BPD uptake by CD45+ leukocytes (Fig. S10), whichrecognize the mAb Fc domain (19). This immune cell uptake isreduced in vitro by covalent attachment of polyethylene glycolpolymer chains to Cet-BPD (2); however, in vivo, we detectedCet-BPD uptake by isolated CD45+ leukocytes in normal tissue(removed from analysis using a size filter; Figs. S2 and S10) andby tumor-infiltrating CD45+ leukocytes (Fig. 3A and Fig. S10).The presence of extensive CD45+ leukocyte tumor infiltratescorroborates preclinical and clinical pathology studies of EOC(39). Histopathologic grading of biopsies matched to micro-endoscope images revealed thin layers of inflammatory cells inmice following treatment (Fig. S6C). Thus, residual tumor in-filtrating and inflammatory leukocytes posttreatment account forthe false-positive cancer cell counts and lowered accuracy ofcancer cell monitoring following treatment. Our data suggest thatintegration of CD45 imaging, using multicolor microendoscopy,

could improve cancer cell-monitoring accuracy by excludingnontumor leukocytes from analysis. However, these cells shouldnot be excluded from quantification of micrometastatic burden.In fact, treatment and monitoring of tumor-associated leukocytesthat uptake and activate Cet-BPD could also prove to be ad-vantageous because these cells have been shown to aid metastaticspread and preparation of the premetastatic niche (40–42).

Note S7: Rationale for the Term “Tumor-Targeted, ActivatablePhotoimmunotherapy.” The dual-function immunoconjugates de-veloped here, for both therapy and imaging, are not only targetedto cancer cells but also require cellular internalization and pro-cessing for activation, thereby improving tumor selectivity. Ly-sosomal activation of fluorescent probes (without any therapeuticcomponent) has been described as “targeted-cancer-cell–specificactivation” (43). However, in reality, other cell types expressthese markers, albeit, normally at a lower level. Here, we showexplicitly that certain nontarget and tumor-associated cells alsoactivate these probes in vivo (Fig. S10 and Note S6). Althoughincreased tumor selectivity is indeed realized, the phrase “cancercell-specific” is misleading. We therefore propose modifying theterminology to specify only what is truly achieved, a “tumor-targeted activation” that enhances selectivity for the tumorbut that does not achieve perfect cancer cell specificity.

Note S8: Limitations of Micrometastasis Burden Imaging withClarifications on the Utility of taPIT Independent from TumorVisualization. Although comprehensive volumetric in vivo mi-croscopy is possible for some organs (44–46), scanning the entirebody in microscopic detail is impractical. Therefore, the potentialvalue of fluorescence microendoscopy—independent of taPIT—is that it enables minimally invasive recognition and monitoring ofmicroscopic residual disease in common sites of recurrence,which will help design therapies (including taPIT and othertreatment modalities) and guide clinical decisions such as con-tinuation of therapy or commencement of salvage therapies. Thisapplication is similar to the present use of laparoscopy for ovariancancer, where clinicians scan the peritoneal cavity for residualdisease. In contrast to laparoscopy, which is an invasive surgicalprocedure with poor resolution and contrast for residual disease,fluorescence microendoscopy of activatable immunoconjugates isminimally invasive and enables accurate visualization of micro-scopic tumor deposits.However, taPIT is a viable andmeaningful therapy independent

of fluorescence microendoscopy and micrometastasis imaging—itis not a “see-first–then-treat” approach. The residual drug-re-sistant disease that leads to mortality responds to PIT, and—dueto the enhanced tumor selectivity achieved by tumor-targetedactivation—taPIT can be applied safely for wide-field treatmentof regions within the body suspected to harbor residual diseasewithout the need of seeing the residual microscopic disease. Asdemonstrated in this study, taPIT of larger regions of tissue canbe accomplished using diffusing tip fiber optics and scatteringmedia to spread out the laser light (Fig. 1F) to treat micro-metastases throughout the entire peritoneal cavity.

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31. Duska LR, Hamblin MR, Miller JL, Hasan T (1999) Combination photoimmunotherapyand cisplatin: Effects on human ovarian cancer ex vivo. J Natl Cancer Inst 91(18):1557–1563.

32. Kessel D (2006) Death pathways associated with photodynamic therapy. Med LaserAppl 21(4):219–224.

33. Chang SK, Rizvi I, Solban N, Hasan T (2008) In vivo optical molecular imaging ofvascular endothelial growth factor for monitoring cancer treatment. Clin Cancer Res14(13):4146–4153.

34. Verma S, Watt GM, Mai Z, Hasan T (2007) Strategies for enhanced photodynamictherapy effects. Photochem Photobiol 83(5):996–1005.

35. de Graeff P, et al. (2009) Modest effect of p53, EGFR and HER-2/neu on prognosis inepithelial ovarian cancer: A meta-analysis. Br J Cancer 101(1):149–159.

36. Valeur B, Berberan-Santos MN (2012) Molecular Fluorescence: Principles andApplications (Wiley-VCH, Weinheim, Germany), 2nd Ed.

37. Savellano MD, Hasan T (2005) Photochemical targeting of epidermal growth factorreceptor: A mechanistic study. Clin Cancer Res 11(4):1658–1668.

38. Aveline BM, Hasan T, Redmond RW (1995) The effects of aggregation, protein binding andcellular incorporation on the photophysical properties of benzoporphyrin derivativemonoacid ring A (BPDMA). J Photochem Photobiol B 30(2-3):161–169.

39. Leinster DA, et al. (2012) The peritoneal tumour microenvironment of high-gradeserous ovarian cancer. J Pathol 227(2):136–145.

40. Kaplan RN, Rafii S, Lyden D (2006) Preparing the “soil”: The premetastatic niche.Cancer Res 66(23):11089–11093.

41. Joyce JA, Pollard JW (2009) Microenvironmental regulation of metastasis. Nat RevCancer 9(4):239–252.

42. Hanahan D, Weinberg RA (2011) Hallmarks of cancer: The next generation. Cell144(5):646–674.

43. Kobayashi H, Choyke PL (2011) Target-cancer-cell-specific activatable fluorescenceimaging probes: Rational design and in vivo applications. Acc Chem Res 44(2):83–90.

44. Yun SH, et al. (2006) Comprehensive volumetric optical microscopy in vivo. Nat Med12(12):1429–1433.

45. Kim P, et al. (2010) In vivo wide-area cellular imaging by side-view endomicroscopy.Nat Methods 7(4):303–305.

46. Gora MJ, et al. (2013) Tethered capsule endomicroscopy enables less invasive imagingof gastrointestinal tract microstructure. Nat Med 19(2):238–240.



High-contrast displaynon-linear scale (saturation)

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Fig. S1. Saturated, nonlinear fluorescence intensity display appears to show high contrast. The image panels from Fig. 1E are shown here as an example. Forquantitative and high-fidelity display, all images within the paper are shown on a linear, nonsaturated scale that is clearly visible on screen but may not bevisible on low-quality prints.



Gaussian convolutionnoise filter

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Itumor,sum (t) = Itumor (x, y, t) PKcontrol (t ti )i

PKtumor ( t f )PKtumor (t ti )

ix,y

Fig. S2. Longitudinal micrometastatic burden quantification by in vivo fluorescence microendoscopy using an image analysis workflow that integratesmultiple experiments to inform batch processing of the data. The workflow holds promise for development into a bioimage informatics platform (1, 2), byadoption of a relational database to facilitate hypothesis-driven, unbiased, and quantitative analyses of in vivo microendoscopy experiments for optimizingtumor recognition accuracy, drug screening, and therapeutic regimen development. (A) Raw images from many experiments. (B) Corresponding backgroundand calibration images of water and 1 μM verteporfin in PBS with 2% Triton X-100, respectively, were collected for each experiment to subtract background(e.g., detector dark current) and to normalize images to a standard for global comparison while also correcting the excitation light profile. (C) A binary maskselects pixels within the coherent fiber bundle for analysis using a 5% maximum intensity threshold, followed by an “erode” operation to avoid including thefiber edges. (D) Mean autofluorescence was calculated for each tissue site (n = 3 mice, 118 fields including no-tumor control and EOC mice): 0.039, peritonealwall; 0.000, pelvic omentum. (E) Mean autofluorescence subtraction was performed for all images. (F) Fits to a simple pharmacokinetic (PK) model (SI Text)were carried out for control no-tumor and EOC mice to facilitate corrections for residual Cet-BPD during multi-immunoconjugate injection experiments. (G)Adjustment of the 99.5% background-rejecting, tumor recognition intensity threshold based on Cet-BPD pharmacokinetics (PK) in nontumor tissue. (H) Se-lection of tumor objects in images using the tumor recognition threshold. (I) Removal of any tumor objects less than 30 μm in dimension based on ROC analysis(Fig. 3 and Fig. S10). (J) Calculation of the integrated tumor fluorescence intensity as a surrogate for micrometastatic burden. The second term subtracts theincreased nonspecific fluorescence background following multiple immunoconjugate injections based on Cet-BPD pharmacokinetics (PK). The final term adjuststhe tumor fluorescence, based on pharmacokinetic (PK) measurements, for increasing tumor signal following multiple immunoconjugate injections.

1. Swedlow JR, Goldberg I, Brauner E, Sorger PK (2003) Informatics and quantitative analysis in biological imaging. Science 300(5616):100–102.2. Millard BL, Niepel M, Menden MP, Muhlich JL, Sorger PK (2011) Adaptive informatics for multifactorial and high-content biological data. Nat Methods 8(6):487–493.



AutofluorescenceCK8, EOCCK8, no tumor control

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In vivo microendosocpy

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Fig. S3. Quantitative intensity thresholds for tumor recognition, ROC analysis, and colocalization analysis. The intensity thresholds reject >95% of pixelscontaining solely nonspecific background fluorescence (solid vertical lines). Frequency counts represent binning of individual pixels (n ≥ 3 mice and numerousfields per mouse for each condition). (A) (Left) Exemplary intensity threshold for rejecting 99.5% of nonspecific Cet-BPD signal following a single injection.(Right) Validation of pharmacokinetics-informed intensity thresholds for multiple Cet-BPD injections. The pharmacokinetics-informed threshold rejects 96.2%of the background fluorescence from normal tissue in control no-tumor mice that received Cet-BPD injections at 24 and 192 h, and chemotherapy at 144 h,before microendoscopy at 216 h (i.e., 24 h after the final Cet-BPD injection). Mean autofluorescence was subtracted before computing the histograms. (B andC) Intensity thresholds for rejecting 99.5% of autofluorescence (Cet-BPD, CD45, and CD31) or no-tumor control (nonspecific) fluorescence (CK8). Thresholdswere determined for both the ROC (B) and 3D colocalization analyses (C).



Human Mouse

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Fig. S4. Cet-BPD selectively binds both human and mouse EGFR, and validation of custom immunofluorescence stains. (A) Human and mouse cell lines werestained with fluorophore-conjugated anti-human and -mouse EGFR mAb (green), anti-human CK8 mAb (orange), anti-mouse CD45 mAb (cyan), and Cet-BPD(1:7) (red). CT26 cells (mouse colorectal carcinoma) were included as a mouse EGFR-expressing cell line (1, 2). EGFR-overexpressing cell lines, including A431(human epidermoid carcinoma), OVCAR5 (human EOC), and CT26 stained brightly with anti-EGFR mAb and Cet-BPD, whereas a low–EGFR-expressing line,T47D, did not. Both A431 and OVCAR5 cells stained positively with anti-CK8 mAb, which targets human CK8. We were inspired to develop the anti-CK8immunostain based on successful clinical reports of cytokeratin staining to selectively image and classify epithelial cancers (3) and micrometastases (4). The anti-human cytokeratin stain applied here is widely applicable to mouse xenograft models of epithelial cancers, for which it has dual selectivity for human epithelialcancer cells. Only J774 cells (mouse macrophages) stained positively with anti-CD45 mAb, which targets immune cells (i.e., leukocytes). J774 cells (CD45+

macrophages) exhibited nonspecific basal uptake of anti-EGFR, anti-CK8, and Cet-BPD. A 100× excess of cetuximab outcompetes Cet-BPD binding to bothhuman and mouse EGFR, as evidenced by very low BPD fluorescence in A431, OVCAR5, and CT26 cells. A 100× excess of trastuzumab, which binds another EGFRfamily member (HER2), does not affect Cet-BPD fluorescence in A431, OVCAR5, or CT26 cells. These results demonstrate highly specific Cet-BPD binding to bothhuman and mouse EGFR (n ≥ 3 images from independent wells of a multiwell plate for each antibody stain and cell line condition) and corroborate a priorreport of cetuximab binding to mouse EGFR (2). (Scale bars: 50 μm.) (B) No significant difference in the ratios (mean ± SEM) of Cet-BPD to anti-EGFR mAbfluorescence was detected among A431, OVCAR5, and CT26 cells (n = 5 images from independent wells per cell line). Background fluorescence was subtractedfrom Cet-BPD and anti-EGFR mAb fluorescence before calculating ratios. By accounting for variations in EGFR expression among the different cell lines, theseresults further confirm the specificity of Cet-BPD binding to mouse and human EGFR, as well as Cet-BPD internalization and activation by both mouse andhuman cells overexpressing EGFR.

1. Cai W, et al. (2007) Quantitative PET of EGFR expression in xenograft-bearing mice using 64Cu-labeled cetuximab, a chimeric anti-EGFR monoclonal antibody. Eur J Nucl Med MolImaging 34(6):850–858.

2. van Houdt WJ, et al. (2010) Oncogenic KRAS desensitizes colorectal tumor cells to epidermal growth factor receptor inhibition and activation. Neoplasia 12(6):443–452.3. Chu P, Wu E, Weiss LM (2000) Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases. Mod Pathol 13(9):962–972.4. Braun S, et al. (2000) Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer. N Engl J Med 342(8):525–533.



No tumor control Disseminated tumorBPD

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Fig. S5. Spectrally unmixed BPD fluorescence (red) and autofluorescence (gray scale) from hyperspectral fluorescence images of the peritoneal cavity (zoomviews of the bowel are shown in Fig. 2C) after verteporfin or Cet-BPD(1:7) administration. Intensity scales are matched for no-tumor control and EOC mice. Theasterisks and the dotted lines demark the peritoneal wall and the bowel, respectively, in each image.



A

0 J cm-1 25 J cm-1

50 J cm-1 75 J cm-1

B C

Fig. S6. Histological evidence that high-dose taPIT overcomes bowel phototoxicity while destroying micrometastases. (A) Histopathologic examination ofH&E-stained bowel tissue sections by a pathologist (E.O.), blinded to the treatment groups, did not indicate any bowel damage. Bowel tissue sections werecollected 7 d after treatment for mice receiving Cet-BPD (1:7; no light, or 0 J·cm−1; 2 mg·kg−1 BPD; n = 1 mouse), as a negative control, or various doses of taPITwith Cet-BPD (1:7; 25–75 J·cm−1 per peritoneal quadrant; 2 mg·kg−1 BPD; n = 3 mice). (Scale bars: 200 μm.) Generally, when bowel phototoxicity is present, it isfatal as well as readily evident during necropsy and by histological evaluation. As a few examples, mortality due to bowel phototoxicity and bowel tissueedema during necropsy were observed for mice that received always-on verteporfin–photodynamic therapy (PDT) in a prior study at elevated photodynamicdoses (1); and Major et al. (2) reported unmistakable histological features of bowel phototoxicity that appear within 3 d of high-dose photodynamic therapy.Corroborating the histopathology results, I.R. performed necropsies blinded to treatment group, including seven mice that received taPIT (Cet-BPD, 1:7; 2 mg/kg BPD; 50 J·cm−1 per peritoneal quadrant with 0% mortality due to taPIT; Fig. 5A). These mice appeared normal other than the presence of metastaticepithelial ovarian cancer and were killed to perform tumor weight evaluation. There were no apparent signs of bowel tissue edema, described above. (B and C)Histopathologic examination of microendoscopy-guided punch biopsies by a pathologist (E.O.), blinded to the treatment groups, suggested a lack of residualtumor nodules in treated mice (n = 6 mice; 6 biopsies total). The pathologist did identify residual inflammatory cells in some of the treated mice. In contrast,multicellular tumor nodules were identified in the majority of biopsies from untreated EOC mice (n = 3 mice, 12 biopsies total; Fig. 6A). An exemplary H&E-stained section is shown for an untreated tumor (B) for comparison with a section from an EOC mouse that received two cycles of taPIT (C) with Cet-BPD (1:7; 2mg·kg−1 BPD; 50 J·cm−1 per peritoneal quadrant). Both of these biopsies appeared to contain tumor nodules based on their coregistered, in vivo fluorescencemicroendoscope images with signal above the fluorescence intensity threshold shown in Fig. 6A, suggestive of the presence of tumor; i.e., we attempted toidentify residual tumor in the taPIT mice using microendoscopy guidance. However, multicellular tumor nodules are evident only in the untreated tumorbiopsy (B). In contrast, the taPIT biopsy contains residual inflammatory cells (Note S6 and Fig. S10) (C). [Scale bars: 1 mm (for image mosaics) and 100 μm (for

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Insets).] Note that, at the treatment course endpoints investigated here, the necrotic tumor cells have been cleared. Our previous work has shown directhistological evidence of tumor cell apoptosis and necrosis by PDT (3) and PIT (4), which are clearly visible at short time points following treatment (<72 h). Thefocus of this study was to monitor the time evolution of micrometastasis response in vivo using fluorescence microendoscopy (validated by qRT-PCR, Fig. 5C,and by H&E histopathology, Fig. 6). Microendoscopy provides direct evidence of longitudinal tumor reduction (Fig. 5E).

1. Molpus KL, et al. (1996) Intraperitoneal photodynamic therapy of human epithelial ovarian carcinomatosis in a xenograft murine model. Cancer Res 56(5):1075–1082.2. Major AL, et al. (2002) Intraperitoneal photodynamic therapy in the Fischer 344 rat using 5-aminolevulinic acid and violet laser light: A toxicity study. J Photochem Photobiol B 66(2):

107–114.3. Chen B, Pogue BW, Hoopes PJ, Hasan T (2005) Combining vascular and cellular targeting regimens enhances the efficacy of photodynamic therapy. Int J Radiat Oncol Biol Phys 61(4):

1216–1226.4. Molpus KL, Hamblin MR, Rizvi I, Hasan T (2000) Intraperitoneal photoimmunotherapy of ovarian carcinoma xenografts in nude mice using charged photoimmunoconjugates. Gynecol

Oncol 76(3):397–404.

A B EOC EC

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Fig. S7. Three-dimensional colocalization statistical analysis of Cet-BPD internalization and activation within epithelial ovarian cancer (EOC) cells in vivo. Theimages are of freshly excised tissue from EOC mice treated in vivo with Cet-BPD(1:7) after application of custom immunostains to visualize EOC cells andmicrovessels [endothelial cells (EC)]. (A) Confocal image mosaic of freshly excised peritoneal wall from an EOC mouse. (B) In similarity to Fig. 3, representativeimages of EOC cells and ECs are shown, as well as the corresponding total, EOC-colocalized, and non–EOC-colocalized Cet-BPD fluorescence. An exemplaryrandomly scrambled Cet-BPD image is also shown (Cet-BPDscr). (C) Cet-BPD scrambling does not significantly alter colocalization with ECs, demonstrating thatEC colocalization is due to random fluctuations. In contrast, Cet-BPD channel scrambling significantly impacts colocalization with EOC cells, which indicatesa nonrandom colocalization. Results are mean ± SEM, n = 7 mice (262 optical sections, 200 random scrambling permutations per optical section, **P < 0.01,****P < 0.0001, two-tailed unpaired t test). [Scale bars: 100 μm (A and B)].



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Fig. S8. In vivo longitudinal microendoscopy and extraction studies of verteporfin pharmacokinetics, and in vivo microendoscopy of nonspecific controlimmunoconjugates (IgG-BPD and Tra-BPD) in control no-tumor and EOC mice. (A) Representative in vivo microendoscopy images from select time points. (Scalebars: 100 μm.) (B) Means (± SEM) pharmacokinetic traces by microendoscopy and extraction averaged over several tissue sites. The trend lines are fits toa simple pharmacokinetic model (SI Text). Microendoscopy verteporfin control, n = 2 mice; tumor, n = 4 mice. Extraction verteporfin control, n = 5 mice; tumor,n = 5 mice (*P < 0.05, ****P < 0.0001, unpaired, two-tailed Student t test). In vivo microendoscopy confirms that verteporfin has tumor selectivity in theperitoneal wall and pelvic omentum as seen in Fig. S5; however, the tumor selectivity is poor in other sites such as the bowel (Fig. 2 C and D and Fig. S4). Notethat the extraction data have a higher signal compared with microendoscopy (which includes only the peritoneal wall and pelvic omentum) in control no-tumor mice due to the inclusion of higher nonspecific accumulation of verteporfin in the bowel and other peritoneal organs (the spleen, liver, and kidney) inthe extraction data. (C) Pharmacokinetic traces (mean ± SEM) for the nonspecific IgG-BPD(1:7) and Tra-BPD(1:5) immunoconjugate controls (Cet-BPD is shownfor comparison). The control no-tumor pharmacokinetic traces (Upper) show that both IgG-BPD and Tra-BPD have increased tissue uptake and retentioncompared with Cet-BPD, likely due to differences in net charge and local charge distributions that strongly influence antibody pharmacokinetics (1). Micro-endoscopy Cet-BPD control, n = 4 mice; tumor, n = 4 mice. Microendoscopy IgG-BPD control, n = 4 mice; tumor, n = 4 mice. Microendoscopy Tra-BPD control,n = 3 mice; tumor, n = 4 mice. (*P < 0.05, **P < 0.01, ***P < 0.001, unpaired, two-tailed Student t test). Increased positive charge tends to increase tissuesequestration and retention (due to heparin sulfate and other anionic proteoglycans on cell surfaces and within the extracellular matrix) (1). Polyclonal IgG isknown to contain isoforms with higher isoelectric points (pI) (pI = 7–9); i.e., increased net positive charge (2). Tra also has a relatively high pI (pI = 9.2) (2)compared with typical pIs for antibodies (pI = 8.4) (1). We measured Cet to have a negative ζ potential (−30 mV; Malvern Zetasizer Nano) in agreement witha prior study that found surface conjugation of Cet to decrease the ζ potential of nanoparticles (3). In contrast, surface conjugation of Tra increases ζ potential(4), presumably due to its net cationic charge. The pharmacokinetic data show that Cet-BPD has the lowest propensity to accumulate in tissue while achievinghigh tumor activation. The approximately twofold enhancement in tumor selectivity (Fig. 4D) for Cet-BPD (EGFR-targeted mAb, moderate EOC expression)versus nonspecific IgG-BPD (nontargeted, polyclonal antibody) and Tra-BPD (HER2-targeted, low EOC expression) agrees with a prior report (5) that also found

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a twofold enhancement for tumor-targeted versus nonspecific antibodies delivered i.p. However, here, we targeted disseminated micrometastases and used anactivatable construct, whereas the other study used large, solid tumors within the peritoneal cavity and radiolabeled antibodies.

1. Boswell CA, et al. (2010) Effects of charge on antibody tissue distribution and pharmacokinetics. Bioconjug Chem 21(12):2153–2163.2. Wiig H, Gyenge CC, Tenstad O (2005) The interstitial distribution of macromolecules in rat tumours is influenced by the negatively charged matrix components. J Physiol 567(Pt 2):

557–567.3. Puvanakrishnan P, et al. (2012) Narrow band imaging of squamous cell carcinoma tumors using topically delivered anti-EGFR antibody conjugated gold nanorods. Lasers Surg Med

44(4):310–317.4. Barua S, et al. (2013) Particle shape enhances specificity of antibody-displaying nanoparticles. Proc Natl Acad Sci USA 110(9):3270–3275.5. Wahl RL, et al. (1988) The intraperitoneal delivery of radiolabeled monoclonal antibodies: Studies on the regional delivery advantage. Cancer Immunol Immunother 26(3):187–201.



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Fig. S9. Validation of qRT-PCR–based micrometastatic burden assessment, and correlation of micrometastatic burden in 1-cm2 biopsies of the peritoneal wall versusthe entire cavity; and treatment regimen and tumor response monitoring time courses, including timelines for Cet-BPD injection, microendoscopy, and treatments. (A)Validation of qRT-PCR–based quantification of micrometastatic burden (total number of EOC cells) by implanting known numbers of EOC cells in the peritoneumfollowed by immediate evaluation (Pearson correlation r = 0.99, n = 5 mice, P = 0.0006). (B) Correlation of tumor burden found in small peritoneal biopsies with thetumor burden found in the entire peritoneal cavity of the samemouse (Spearman r = 0.76, n = 48mice, P < 0.0001). Three data points are at the origin (0, 0), and someoutliers are present with 0 EOC cells detected in the biopsy, whereas many EOC cells were found in the entire cavity. This suggests that, as the tumor is destroyed, smallbiopsies cannot report on the overall tumor burden and necessitates the use of in vivo imaging for “optical biopsy” throughout the cavity. (C) Timeline for two cyclesof taPIT. (D and E) Timelines for one to two cycles of taPIT combined with combination chemotherapy. Cet-BPD (no light) treatments were performed on identicalschedules. All microendoscopy was done 8–24 h after Cet-BPD injection. “No treatment” mice received only one Cet-BPD injection, 24 h before the endpoint.



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Fig. S10. Spatial filter to reject single-cell objects, which are primarily peritoneal CD45+ immune cells (IC) that take up Cet-BPD in normal tissues. False-positive, single-cell objects in normal and EOC peritoneal tissue were identified as CD45+ leukocytes, which include Fc receptor-expressing monocytes (e.g.,macrophages) known to uptake antibodies nonspecifically (1). (A) Immune cells (ICs) (i.e., CD45+ leukocytes) are frequently encountered in the peritoneum andinfiltrate peritoneal micrometastases, composing more than 50% of the tumor mass (2). (B) Enlarged views of the regions indicated in A, where 30-μm di-mension circles are overlaid on single-cell objects. [Scale bars: 1 mm (A) and 30 μm (B).]

1. Savellano MD, Hasan T (2003) Targeting cells that overexpress the epidermal growth factor receptor with polyethylene glycolated BPD verteporfin photosensitizer immunoconjugates.Photochem Photobiol 77(4):431–439.

2. Leinster DA, et al. (2012) The peritoneal tumour microenvironment of high-grade serous ovarian cancer. J Pathol 227(2):136–145.

Movie S1. In vivo fluorescence microendoscopy of a no-tumor control mouse. The movie shows low Cet-BPD fluorescence in the absence of tumor. A fewisolated bright cells are apparent in the normal tissue, which were excluded from analysis using a spatial filter (Fig. S10).

Movie S1


http://www.pnas.org/lookup/suppl/doi:10.1073/pnas.1319493111/-/DCSupplemental/sm01.mov


Movie S2. In vivo fluorescence microendoscopy of an untreated epithelial ovarian cancer mouse. The movie highlights bright Cet-BPD fluorescence in thepresence of extensive micrometastatic disease in an untreated mouse.

Movie S2

Movie S3. In vivo fluorescence microendoscopy of an epithelial ovarian cancer mouse that received Cet-BPD (no light) treatment. The movie indicates lowtreatment efficacy of Cet-BPD in the absence of near-infrared photoactivation.

Movie S3





Movie S4. In vivo fluorescence microendoscopy of an epithelial ovarian cancer mouse that received taPIT. The movie demonstrates micrometastatic burdenreduction by near-infrared photoactivation of Cet-BPD.

Movie S4




supporting information - pnas...2014/02/26 · at a concentration of 20 μg/ml, and anti-ck8,...

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