Using Atomic Force Microscopy to Predict Tumor Specificity of ICAM1

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Using Atomic Force Microscopy to Predict Tumor Specificity of ICAM1 Antibody-directed Nanomedicines Peng Guo, Biran Wang, Daxing Liu, Jiang Yang, Kriti Subramanyam, Craig McCarthy, Jacob Hebert, Marsha A. Moses, and Debra Auguste Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.7b04801 • Publication Date (Web): 05 Mar 2018 Downloaded from http://pubs.acs.org on March 6, 2018

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Title Using Atomic Force Microscopy to Predict Tumor Specificity of ICAM1 Antibodydirected Nanomedicines Authors P. Guo,1,2,3 B. Wang,1ǁ D. Liu,1,5 J. Yang,2,3 K. Subramanyam,4§ C. R. McCarthy,1 J. Hebert,5 M. A. Moses,2,3† and D. T. Auguste1,5†* Affiliations 1. Department of Biomedical Engineering, The City College of New York, 160 Convent Avenue, New York, NY 10031, United States 2. Vascular Biology Program, Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, United States 3. Department of Surgery, Harvard Medical School and Boston Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, United States 4. School of Engineering and Applied Sciences, Harvard University, 29 Oxford Street, Cambridge, MA 02115, United States 5. Department of Chemical Engineering, Northeastern University, 360 Huntington Avenue, Boston, MA 02115, United States † These authors contribute equally and are co-last authors. *Corresponding author. Email: [email protected]; Tel: +1-617-285-6279; Fax: +1-617-373-2209 ǁ Current address: Department of Industrial & Systems Engineering, Texas A&M University, 101 Bizzell Street, College Station, TX 77843, United States § Current address: Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, 500 Main Street, Cambridge, MA 02139, United States

Keywords: AFM, ICAM1, TNBC, liposomes, drug delivery, nanomedicine

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Abstract Atomic force microscopy (AFM) is a powerful tool to detect in vitro antibody-antigen interactions. To date, however, AFM-measured antibody-antigen interactions have yet to be exploited to predict in vivo tumor specificity of antibody-directed nanomedicines. In this study, we have utilized AFM to directly measure the biomechanical interaction between live triple negative breast cancer (TNBC) cells and an antibody against ICAM1, a recently identified TNBC target. For the first time, we provide proof-of-principle evidence that in vitro TNBC cellICAM1 antibody binding force measured by AFM on live cells more precisely correlates with in vivo tumor accumulation and therapeutic efficacy of ICAM1 antibody-directed liposomes than ICAM1 gene and surface protein overexpression levels. These studies demonstrate that live cellantibody binding force measurements may be used as a novel in vitro metric for predicting the in vivo tumor recognition of antibody-directed nanomedicines.

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Introduction Tumor-targeting activity of nanomedicines is often governed by specific antibodyantigen or ligand-receptor interactions between drug delivery systems and cancer cells. For example, antibody-directed targeting is commonly used to preferentially accumulate nanomedicines in tumor sites1–3. It requires a tumor-specific antibody or ligand to be conjugated to a drug delivery system to recognize and bind antigen on receptor-overexpressing tumors. To date, MM-302 (human epidermal growth factor receptor 2 (HER2) antibody-conjugated liposomal doxorubicin), an anti-cancer liposome, has demonstrated promising clinical benefits for HER2-positive metastatic breast cancer patients by significantly improving median progression free survival by 7.6 months with an overall response rate of 11%4. However, unlike HER2-positive breast cancer, no clinically effective therapeutic target has been identified for TNBC, a highly malignant form of breast cancer defined by the absence of HER2, estrogen receptor (ER), and progesterone receptor (PR). Identification of a TNBC target therefore is pivotal for the development of tumor-targeted therapeutics and subsequent positive patient prognosis5–9. Most tumor-targeting studies focus on the identification of overexpressed genes or proteins in cancer cells. A central question is whether these overexpressed genes/proteins can be used to effectively recognize and target primary tumors and metastatic lesions and, in turn, improve therapeutic efficacy. However, quantifying the overexpression of gene/protein in cancer cells alone may not be sufficient to answer this question, given that overexpression does not always translate into specific targeting in vivo. The localization, organization, and ligand binding strength of a molecular target may play critical roles in modulating tumor recognition and targeting. Acquisition of this information is often limited by conventional assays that evaluate the average levels of a target (e.g., PCR, western blot) or that probe the ligand-target interaction in the absence of living cells (e.g. immunoprecipitation). We have previously demonstrated that AFM is a powerful tool to directly detect antibody-antigen biomechanical interactions on live cell surfaces10,11. To date, however, the correlation between in vitro live cell-antibody binding forces and the in vivo tumor targeting activity of antibody-directed nanomedicine has yet to be exploited. Given its importance for tumor recognition, we hypothesized that the live cell-antibody binding force quantified using AFM in vitro could be used as a quantitative metric to predict the in vivo tumor recognition of its antibody-directed nanomedicine. To test this hypothesis, we used AFM to quantitatively map antibody binding events of ICAM1, a recently discovered TNBC target12,13, on live TNBC and non-neoplastic cell surfaces, and subsequently calculated the TNBC-specific binding force. Using an orthotopic TNBC tumor model, we presented the proof-of-principle evidence that correlates the in vitro TNBC-specific binding force of ICAM1 antibody with the in vivo tumor recognition and therapeutic efficacy of ICAM1 antibody-directed liposomes. To our knowledge, this is the first demonstration of the use of the AFM-measured in vitro live cell-antibody binding force to predict tumor accumulation and efficacy of an antibody-directed nanomedicine and that, in the case studied here, this approach is a more precise metric than gene and protein expression levels.

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Assessment of cell surface antigens overexpressed in TNBC We previously demonstrated that ICAM1 levels are significantly elevated in human TNBC tissues and cell lines, suggesting it as a novel TNBC target12,13. However, because no quantitative comparison between ICAM1 and other reported TNBC targets including epidermal growth factor receptor (EGFR)14, plasminogen activator urokinase receptor (PLAUR)15,16, CD4417 and transferrin receptor (TFRC)18 has been conducted, it remains unknown which cancer target is optimal for TNBC-targeting nanomedicine. In this study, we performed an unbiased and quantitative assessment of a panel of 40 cancer-related cell surface antigens on TNBC cells (Table S1).We quantified protein levels on the surface of human TNBC MDA-MB-231 cells and non-neoplastic MCF10A cells by flow cytometric analysis (Figure 1a). TNBC target candidates were ranked according to their overexpression levels on MDA-MB-231 cells relative to MCF10A cells. Twenty-two of 40 examined antigens were upregulated on MDA-MB-231 cells; the top ten TNBC-overexpressed antigens are listed in Figure 1b. ICAM1emerged as the most significantly overexpressed molecule, with respect to the control, among the 40 tested candidates. ICAM1 protein was expressed at a level that is 46.4-fold higher on MDA-MB-231 cells than MCF10A cells. We further compared the cell surface densities of the top ten TNBCoverexpressed antigens on non-neoplastic MCF10A cells (Figure 1c). ICAM1 was expressed at a significantly lower level on MCF10A cells relative to other highly overexpressed TNBC targets such as integrin alpha 3(ITGA3) and integrin beta 1(ITGB1). TFRC and CD44, two broadlyused cancer targets in nanomedicine, were identified as being unsuitable for TNBC-targeting due to their high expression on non-neoplastic MCF10A cells (Figure 1a). Given its tumor specificity and overexpression levels, we postulated that ICAM1 is a key target for TNBC-targeted nanomedicine, and we chose to focus on ICAM1 to investigate its live cell-antibody biomechanical interactions on human TNBC cells and its implications for in vivo TNBC-targeted drug delivery. It is worth noting that the gene and surface protein overexpression levels of ICAM1 on MDA-MB-231 cells, two established quantitative metrics for defining cancer targets, are 13.912and 46.4-folds over non-neoplastic controls (MCF10A cells), respectively (Figure S1). Direct detection of the TNBC-specific binding force of ICAM1 antibody Although AFM is a commonly used tool to investigate antibody-antigen interactions, most studies are performed at the protein level in the absence of living cells. Our lab has pioneered the use of AFM to investigate live cell-ligand interactions for cancer target identification10,11. In this study, we utilized AFM to quantitatively probe the biomechanical interactions between live human TNBC cells (MDA-MB-231) and ICAM1 antibody. We further compared these results with non-neoplastic human mammary epithelial cells (MCF10A). As shown in Figure 2a, we functionalized the AFM tip with ICAM1 antibodies (1200 ± 300 molecule/µm2), and used this functionalized AFM tip-cantilever assembly to probe the binding forces between the ICAM1antibody attached on the AFM tip and antigens presented on the live cell surface10,19,20. The average binding force was quantified from the difference in the approach and retract curves at the pull-off point21.As shown in Figure 2b and Figure S2, the ICAM1 antibody demonstrated an average binding force of 523 ± 113 pN on live MDA-MB-231 cells, which was significantly higher than that of its non-targeting counterpart IgG (96 ± 10 pN). Because the single antibody-antigen interaction was reported as high as 214 pN21, we postulated that our ICAM1 antibody binding force of 527 pN is due to multiple antibody-antigen interactions between the AFM tip and the cell surface. We further compared the average binding

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forces of the ICAM1 antibody on TNBC MDA-MB-231 cells (523 ± 113 pN, black bar) and non-neoplastic MCF10A cells (336 ± 33 pN, black bar) in Figure 2d, which indicated that the ICAM1 antibody had a stronger adhesion for the MDA-MB-231 cell membrane than the nonneoplastic MCF10A cell membrane. To our surprise, the binding force of ICAM1 antibody on live MDA-MB-231 cells is only 1.6-fold higher than that of non-neoplastic controls (MCF10A cells). It was not our expectation that the TNBC-specific binding force ratio of the ICAM1 antibody (1.6-fold) would be substantially lower than its gene and surface protein overexpression levels (13.9 and 46.4-fold, respectively). However, we later found that this in vitro live cellantibody binding force difference between TNBC and non-neoplastic cells has a determinative role in regulating both in vitro and in vivo tumor recognition of ICAM1 antibody-directed liposomes, which is more precise and efficient as a predictive factor than established gene and surface protein overexpression levels. From these results, we calculated the TNBC-specific binding force of ICAM1 antibody as 187 pN based on the binding force difference between the TNBC cell (523 pN) and non-neoplastic MCF10A cell (336 pN). In addition to the overexpression level, the organization of antigens on the cell membrane is another key factor driving differences in live cell-antibody binding behavior11. AFM-detected adhesive events were also used to spatially map the binding forces on MDA-MB-231 (Figure 2e) and MCF10A (Figure 2f) cell surface. As shown in Figure 2e, this AFM map reveals that ICAM1 molecules were heterogeneously organized on the cell surface and binding force “hotspots” for the ICAM1 antibody were observed on MDA-MB-231 cell membranes (highlighted in Figure 2e). These “hot-spots” are predicted to be the primary binding sites for ICAM1 antibodydirected nanomedicine due to the high binding forces. ICAM1 molecules may be enriched in membrane lipid rafts of MDA-MB-231 cells to facilitate functional signaling22. Lipid rafts, present in cell membranes, are gel-phase domains rich in cholesterol the colocalized cell membrane proteins, affecting antibody-antigen interactions in a cholesterol-dependent manner23,24. In order to determine whether ICAM1 binding forces are dependent on the organization of ICAM1 molecules in lipid rafts on the cell membrane, we performed a blocking experiment using methyl-beta-cyclodextrin (MCD), a cholesterol-depleting drug, to disrupt lipid rafts on MDA-MB-231 and MCF10A cells; we then re-measured the average binding force. As shown in Figure 2c, MCD treatment did not affect the average ICAM1 expression on MDA-MB-231 cell surfaces. Similar results were also observed in other cell types15. While MCD treatment had no obvious effect on ICAM1 cell surface expression, MCD did impede the live cell-ICAM1 antibody interaction by delocalizing cell membrane lipid raft-associated molecules25. In Figure 2e, the "hot spots" of ICAM1 adhesion events disappeared after MCD treatment, and the average adhesion force between the ICAM1 antibody and MDA-MB-231 cells significantly decreased from 523 ± 113 pN to 277 ± 46 pN in the presence of MCD (Figure 2d, grey bars) correlating with disperse adhesion maps (Figures 2e and f). No difference was observed between MCF10A cells treated with or without MCD due to its ICAM1 deficiency (Figures 2d and f). Therefore, the selective and strong ICAM1 antibody binding force with the MDA-MB-231 cell membrane is attributed to both the overexpression and the organization of ICAM1 molecules presented on MDA-MB-231 cell membranes.

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Construction of ICAM1 antibody-directed liposomes We next engineered a series of ICAM1 antibody-directed liposomes to investigate the implications of the in vitro live cell-antibody binding force measurements in TNBC-targeted drug delivery. ICAM1 antibody-conjugated, doxorubicin-encapsulating liposomes (ICAM-DoxLPs) were prepared as a TNBC-specific therapeutic agent, as described in Figure 3a. ICAMDox-LPs were comprised of 95 mol% DOPC and 5 mol% DSPE-PEG-COOH. The PEG chain (2 kDa) in DSPE-PEG-COOH improves liposome circulation time26,27. ICAM1 antibody or nonspecific immunoglobulin G (IgG) was conjugated to the carboxyl terminus of the PEG chain. IgG-conjugated, doxorubicin encapsulating liposomes (IgG-Dox-LPs) were prepared as controls. As-synthesized liposomes are characterized in Table 1. Hydrodynamic diameters of ICAM-DoxLPs and IgG-Dox-LPs were 105 ± 31 and 101 ± 24 nm, respectively, as determined by dynamic light scattering (DLS, Figure 3b). Polydispersity indexes (PDIs) of both liposomes were close to 0.1, demonstrating uniformity. In addition, the zeta potentials of ICAM-Dox-LPs and IgG-DoxLPs were -8.8 ± 6.7 and -4.8 ± 4.1 mV, respectively. We used the transmembrane gradient method to encapsulate Dox in liposomes28. The Dox encapsulation efficiencies of ICAM-DoxLPs (92.0 ± 1.6%) and IgG-Dox-LPs (91.5 ± 0.5%) were comparable. Furthermore, the surface densities of conjugated ICAM1 antibody or non-specific IgG were quantified as 3,040 ± 20 molecules/µm2 for ICAM-Dox-LPs and 3,100 ± 28 molecules/µm2 for IgG-Dox-LPs. This is equivalent to approximately 96 molecules per liposome. We also investigated the storage stability of constructed ICAM-Dox-LPs and found that it maintained its hydrodynamic size in cell culture medium (DMEM) with 10% serum for 28 days without aggregation (Figure 3c). We measured the release profiles of Dox from ICAM-Dox-LPs at pH 7.4 and 5.5 in order to mimic the extra- and intracellular environments, respectively (Figure 3d)29–31 and found ICAM-Dox-LP released its cargo faster at the lower pH. In vitro binding affinity of ICAM1 antibody-directed liposomes We first quantified in vitro TNBC cell binding of ICAM1-directed liposomes by flow cytometry. Liposomes encapsulating rhodamine-dextran (RD, 10 kDa) were used to avoid the cytotoxic effect of doxorubicin. Cellular binding and uptake of the ICAM1 antibody or IgG labeled, RD encapsulating liposomes (ICAM-RD-LPs or IgG-RD-LPs) were assessed on three TNBC cell lines: MDA-MB-231, MDA-MB-436 and MDA-MB-157, in comparison with nonneoplastic MCF10A cells. As shown in Figure 3e, MDA-MB-231, MDA-MB-436 and MDAMB-157 cells demonstrated 2.4, 3.3 and 2.3-fold higher binding of ICAM-RD-LPs in cell culture medium containing 10% serum relative to non-specific IgG-RD-LPs, respectively. No difference in binding and uptake between ICAM-RD-LPs and IgG-RD-LPs was detected on MCF10A cells due to its lack of ICAM1 expression. These findings demonstrate that ICAM1 antibodies covalently conjugated on the surface of ICAM-RD-LPs maintain their activity and selectively recognize TNBC cells via the ICAM1 antibody-antigen interaction. The in vitro TNBC-liposome binding is consistent with the high binding forces measured on TNBC cells relative to MCF10A cells (Figures 2d-f). To assess the TNBC-specific cytotoxicity of ICAM-Dox-LPs, we performed proliferation assays on the three TNBC cell lines treated with ICAM-Dox-LPs as a function of Dox concentration. ICAM-LPs, Free Dox, and IgG-Dox-LPs were selected as controls. In all three TNBC cell lines, ICAM-Dox-LPs demonstrated substantially higher in vitro cytotoxicity in

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comparison to non-specific IgG-Dox-LPs. Half maximal inhibitory concentrations (IC50s) were calculated from the cytotoxicity curves. For MDA-MB-231 cells (Figure 3f), the IC50 of ICAMDox-LP was 6.5µg/mL, approximately 1.8-fold less than the IC50 of IgG-Dox-LP (11.4 µg/mL). Similar trends were observed in the MDA-MB-436 and MDA-MB-157 TNBC cell lines (Figures 3g and h). ICAM-LPs did not exhibit cytotoxicity in TNBC cells. These findings demonstrated that introducing a TNBC specific-binding function to liposomal doxorubicin via the ICAM1 antibody can significantly improve its cytotoxicity to TNBC cells relative to non-specific IgGDox-LPs. In vivo tumor recognition and efficacy of ICAM1 antibody-directed liposomes To determine whether the in vitro TNBC-specific binding force of ICAM1 antibody translates into improved in vivo tumor specificity and therapeutic efficacy, we examined the distribution of ICAM1 antibody-directed liposomes by near-infrared (NIR) fluorescent imaging in an orthotopic TNBC model. MDA-MB-231 cells were orthotopically implanted in nude mice. NIR fluorescent imaging was performed on two groups of tumor-bearing mice injected with either ICAM1 antibody or IgG conjugated liposomes labeled with a NIR dye DiR (ICAM-DiRLPs or IgG-DiR-LPs). Each group was scanned at 4 h, 24 h, and 48 h post injection. The representative images in Figure 4a show that accumulation of ICAM-DiR-LPs was significantly increased at TNBC tumor sites relative to that of non-specific IgG-DiR-LPs. Mice injected with ICAM-DiR-LPs exhibited a 1.2-fold (4 h), a 1.5-fold (24 h), and a 1.6-fold (48 h) increase in tumor-specific fluorescence compared to those injected with IgG-DiR-LPs, suggesting that ICAM-DiR-LPs significantly improved TNBC tumor accumulation by actively targeting the TNBC tumor via live cell-antibody interactions (Figure 4b). The biodistribution of ICAM1 antibody-directed liposomes was evaluated by quantifying ex vivo NIR fluorescent signals in collected organs and tumors. Figures 4c and d show comparative liposome accumulation in six organs (liver, spleen, lung, kidney, brain, and heart) and TNBC tumors harvested from mice at 48 h after a single tail vein administration of IgG-DiRLPs or ICAM-DiR-LPs. Correlating with the in vivo imaging results, the accumulation of ICAMDiR-LPs in TNBC tumors was approximately 1.5-fold higher than that of IgG-DiR-LPs. For the six organs analyzed, liver and spleen were the two primary accumulation sites for both ICAM1targeted and non-specific-IgG liposomes, as observed in other liposome studies32,33 and there was no significant difference observed between ICAM-DiR-LP and IgG-DiR-LP groups. It is noteworthy that the in vivo and ex vivo MDA-MB-231 tumor accumulation of ICAM-DiR-LPs (1.6 and 1.5-fold over IgG-DiR-LP) are consistent with the in vitro TNBC-specific binding force of ICAM1 antibody (1.6-fold over non-neoplastic controls), but not with ICAM1gene and surface protein overexpression levels (13.9 and 46.4-fold over non-neoplastic controls) due to the determinative role of live cell-antibody interaction in tumor recognition. We further examined whether ICAM1 antibody-directed liposomes were able to convert their in vivo TNBC tumor-targeting activity into improved therapeutic efficacy. ICAM-Dox-LPs were injected intravenously into nude mice bearing orthotopic TNBC tumors (MDA-MB-231 cells). PBS and non-targeted IgG-Dox-LPs were also tested as controls. After a 24-day treatment regimen, the administration of ICAM-Dox-LPs efficiently inhibited TNBC tumor growth in comparison with PBS and IgG-Dox-LP groups (Figure 4e). Quantified tumor mass results revealed that ICAM-Dox-LPs significantly inhibited TNBC tumor growth by 66%, equivalent to

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an approximately 1.7-fold increased therapeutic efficacy over IgG-Dox-LP that closely matches the in vitro TNBC-specific binding force (1.6-fold over non-neoplastic cells) and in vivo tumor recognition (1.6-fold over non-targeting liposomes). All groups of mice maintained their body weight without significant loss during these treatment periods (Figure 4f). We performed hematoxylin and eosin (H&E) staining and immunohistochemical staining of ICAM1 on sections of excised TNBC tumors (Figure 4g). High expression levels of ICAM1 were present in TNBC tumors from all three treatment groups (PBS, IgG-Dox-LP, and ICAM-Dox-LP), indicating that the differences in the therapeutic efficacy among the three treatment groups was not due to any difference in ICAM1 levels in tumors. In summary, these results confirmed that ICAM-Dox-LPs rely on their ICAM1 antibody-mediated binding force to specifically recognize ICAM1 overexpressing tumors in vivo and to inhibit tumor growth. Validate the correlation between AFM and in vivo findings using EGFR antibody EGFR is a well-established TNBC target34–36 and is ranked 6th in our top 10 TNBC targets with an overexpression level of 357,000 ± 5,700 molecules/cell, which is approximately 5.6-fold less than ICAM1 (2,010,000 ± 27,000 molecules/cell) (Figure 1B) on the MDA-MB-231 cell surface. In our AFM measurements, the EGFR antibody demonstrated average binding forces of 375 ± 72 pN on live human TNBC MDA-MB-231 cells (Figure S3a) and 307 ± 47 pN on live MCF10A cells (normal controls, Figure S3b). We found that the binding force of the EGFR antibody (375 pN) on MDA-MB-231 cells is significantly lower than that of the ICAM1 antibody (527 pN), probably due to differences in antigen number and organization on MDAMB-231 cells. Meanwhile, both EGFR and ICAM1 antibodies also exhibited similar binding forces (approximately 300 pN) on non-neoplastic MCF10A cells due to the low expression of both antigens. Based on these results, we further calculated the TNBC-specific binding force of the EGFR antibody, which is only 68 pN in comparison with that of the ICAM1 antibody which is 187 pN. These AFM data indicate that the binding force of antibody on live cell surface is positively correlated with its antigen expression and confirmed that the ICAM1 antibody has a stronger affinity toward TNBC cells than the EGFR antibody. To validate its predictive value for in vivo tumor specificity, we engineered near infrared dye, DiR-labelled, EGFR antibody-conjugated liposomes (EGFR-DiR-LPs) and quantified their tumor accumulation in comparison with IgG-DiR-LPs and ICAM1-DiR-LPs in the same orthotopic TNBC tumor model as described above. We plotted TNBC tumor accumulations of IgG-DiR-LP, EGFR-DiR-LP, and ICAM1-DiR-LP versus their TNBC-specific binding forces (Figure S4) and observed a strong positive correlation between the AFM measured binding force and the tumor specificity of antibody-directed nanomedicines regardless of different antibody epitopes. We further learned from this correlation that the TNBC-specific binding force of EGFR antibody (68 pN) is not strong enough to generate a significant increase in tumor accumulation of its antibody-directed liposomes (particle size: approximately 100 nm) in comparison with nontargeting IgG-DiR-LP (same 100 nm particle size). However, when the TNBC-specific binding force increased to 187 pN (ICAM1 antibody), we did observe a significant increase in tumor accumulation of ICAM1 antibody-directed liposomes with the same particle size. We postulated that this tumor specificity can be further optimized by either selecting a targeting molecule with an even higher TNBC binding force or altering the mechanical properties of the nanoscale drug delivery system. This information may be beneficial for the design of future TNBC-targeted nanomedicines. Due to the fact that the EGFR antibody itself has an inhibitory effect on TNBC

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tumor growth34–36 and can mask the effect of TNBC-targeted delivery of doxorubicin by EGFR antibody-directed liposomes, we did not compare the therapeutic efficacy between the EGFR antibody-conjugated, doxorubicin-encapsulating liposomes (EGFR-Dox-LP) and ICAM-DoxLP. Efficient tumor-specific delivery of therapeutics in vivo remains a challenge in nanomedicine research. Here, we report a novel strategy, utilizing AFM, which predicts in vivo tumor recognition of antibody-directed nanomedicines. We directly measured the live cellantibody biomechanical interaction using ICAM1 antibody-functionalized AFM on live TNBC cells in comparison to non-neoplastic human mammary epithelial cells. We used this method to develop a simple and potentially universal metric (“the TNBC-specific binding force”) for predicting the in vivo tumor targeting activity of a nanomedicine. Compared with other established predictive factors (e.g. gene or protein overexpression levels), our proof-of-principle animal studies showed that this AFM-based method provides a more precise and efficient evaluation of tumor-binding events for antibody-directed nanomedicine (Figure 5). Furthermore, the high-resolution imaging feature of AFM enables the spatio-temporal visualization of specific binding sites on live TNBC cell surfaces (Figure 2e and f), providing information on the localization and organization of cell membrane antigen that is critical for live cell-antibody biomechanical interactions. Our findings that the in vitro TNBC-specific binding force correlates with the in vivo TNBC tumor accumulation of ICAM1 antibody-directed liposomes may have direct implications for the design of TNBC-targeted therapeutics.10,11,19,20,37,38Li et al. reported that Rituximab, a FDA-approved CD20 antibody for Non-Hodgkin's Lymphoma (NHL) treatment, exhibited a binding force of 54 ± 34 pN on patient-derived NHL B cells and 21 ± 19 pN on normal red blood cells, indicating a NHL-specific binding force of 33 pN.37 In comparison, we quantified the TNBC tumor-specific binding force of ICAM1 antibody as being 187 pN, which is greater than the NHL-specific binding force of Rituximab. Thus, we reasoned that combining a TNBCspecific ligand e.g., ICAM1 antibody, to clinically-approved nanomedicines (e.g. Doxil or Abraxane), would enable them to more efficiently recognize and target TNBC tumors and metastatic lesions and, in turn, may increase the drug dosage in tumors, reduce non-specific uptake and attenuate adverse side-effects. Our in vivo biodistribution studies using an orthotopic mouse TNBC model validated that the ICAM1 antibody-directed liposomes achieved approximately 80% more accumulation in tumor sites than the non-targeted IgG controls. In summary, we demonstrated that the live cell-antibody binding force data, acquired through AFM, maybe used as a novel metric to predict in vivo tumor recognition of antibodydirected nanomedicines. This AFM method used biomechanic parameters that can be measured on individual cells and is, in principle, applicable to a broad range of tumor-targeting molecules (e.g. natural ligands, engineered peptides or aptamers). Moreover, the application of our methodology in the screening and identification of novel molecular targets may also be extended to multiple cancers.

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Acknowledgments D. Auguste acknowledges the support of NIH 1DP2CA174495. M. Moses acknowledges the support of NIH R01CA185530 and the Breast Cancer Research Foundation. We thank Kristin Johnson of the Vascular Biology Program at Boston Children’s Hospital for assistance with the schematic illustration.

Author contributions P.G., B.W., M.A.M. and D.T.A. were responsible for the experimental design; P.G., B.W., D.L., J.Y., K.S., C.R.M. and J.H. performed the experiments; P.G., B.W., D.L., J.Y., M.A.M. and D.T.A. analyzed data. P.G., B.W., M.A.M. and D.T.A. wrote the paper. Competing interests The authors declare no conflict of interest.

Supporting Information Available: Materials and Methods,supplemental figures S1-S4, supplemental table S1.

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Figures captions Figure 1. Ranking of 40 cancer-related antigens based on their levels on the TNBC cell surface. (a) Comparative flow cytometric analysis of TNBC target candidate protein levels on the surfaces of MDA-MB-231 (TNBC) and control MCF10A (non-neoplastic) cells. (b) Overexpression of the top ten target candidates from panel (a) are quantified for TNBC. The overexpression of each antigen was calculated using the following equation: ܱ‫= ݊݋݅ݏݏ݁ݎ݌ݔ݁ݎ݁ݒ‬ ‫்݊݋݅ݏݏ݁ݎ݌ݔܧ‬ே஻஼ − ‫݊݋݅ݏݏ݁ݎ݌ݔܧ‬ே௢௡ି௡௘௢௣௟௔௦௧௜௖ (molecules/cell). Two-tailed p value was calculated on the basis of surface expression difference between MDA-MB-231 and MCF10A cells. All ten targets were significantly overexpressed in TNBC cells compared to the control. (c) Expression of the top ten target candidates on cell surface of non-neoplastic MCF10A cells. ). The mean values and error bars are defined as mean and S.D., respectively Figure 2. AFM measurement of live cell-antibody biomechanical interaction. (a) Schematic illustration of AFM probing ICAM1 antibody binding force on live human MDA-MB-231 (TNBC) and MCF10A (non-neoplastic) cells. (b) Binding force of ICAM1 antibody or nonspecific IgG with MDA-MB-231 cell membrane was detected by AFM using ICAM1 antibody or IgG functionalized AFM tip. (c) Flow cytometric analysis of ICAM1 expression on the MDAMB-231 cell surface pre- and post-MCD treatment. (d) Binding force of ICAM1 antibody to MDA-MB-231 and MCF10A cells probed with ICAM1 antibody functionalized AFM cantilevers pre- and post-MCD treatment. (e and f) Adhesion maps of ICAM1 antibody binding events on live MDA-MB-231 (e) and MCF10A (f) cell membrane pre- and post-MCD treatment. (The dashed cycles illustrate the area subjected to high ICAM1 adhesive events). (* p