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Supplementary Data from Macrophage-Targeted Therapy Unlocks Antitumoral Cross-talk between IFNγ-Secreting Lymphocytes and IL12-Producing Dendritic Cells

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R− cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor–mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell–targeted immunotherapies and promote antitumor immunity.See related Spotlight by Burrello and de Visser, p. 4.

The heterogeneous delivery of drugs in tumors is an established process contributing to variability in treatment outcome. Despite the general acceptance of variable delivery, the study of the underlying causes is challenging, given the complex tumor microenvironment including intra- and intertumor heterogeneity. The difficulty in studying this distribution is even more significant for small-molecule drugs where radiolabeled compounds or mass spectrometry detection lack the spatial and temporal resolution required to quantify the kinetics of drug distribution in vivo. In this work, we take advantage of the synthesis of fluorescent drug conjugates that retain their target binding but are designed with different physiochemical and thus pharmacokinetic properties. Using these probes, we followed the drug distribution in cell culture and tumor xenografts with temporal resolution of seconds and subcellular spatial resolution. These measurements, including in vivo permeability of small-molecule drugs, can be used directly in predictive pharmacokinetic models for the design of therapeutics and companion imaging agents as demonstrated by a finite element model. Mol Cancer Ther; 13(4); 986–95. ©2014 AACR.

PDF file - 401KB, Supplemental file containing a mathematical model description, Figure S1 showing the modeling approach, Table S1 with model parameters, and Figure S2 graphing cellular clearance.

Supplementary Video 1 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 1 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 2 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 2 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Cancer relies upon frequent or abnormal cell division, but how the tumor microenvironment affects mitotic processes in vivo remains unclear, largely due to the technical challenges of optical access, spatial resolution, and motion. We developed high-resolution in vivo microscopy methods to visualize mitosis in a murine xenograft model of human cancer. Using these methods, we determined whether the single-cell response to the antimitotic drug paclitaxel (Ptx) was the same in tumors as in cell culture, observed the impact of Ptx on the tumor response as a whole, and evaluated the single-cell pharmacodynamics (PD) of Ptx (by in vivo PD microscopy). Mitotic initiation was generally less frequent in tumors than in cell culture, but subsequently it proceeded normally. Ptx treatment caused spindle assembly defects and mitotic arrest, followed by slippage from mitotic arrest, multinucleation, and apoptosis. Compared with cell culture, the peak mitotic index in tumors exposed to Ptx was lower and the tumor cells survived longer after mitotic arrest, becoming multinucleated rather than dying directly from mitotic arrest. Thus, the tumor microenvironment was much less proapoptotic than cell culture. The morphologies associated with mitotic arrest were dose and time dependent, thereby providing a semiquantitative, single-cell measure of PD. Although many tumor cells did not progress through Ptx-induced mitotic arrest, tumor significantly regressed in the model. Our findings show that in vivo microscopy offers a useful tool to visualize mitosis during tumor progression, drug responses, and cell fate at the single-cell level. Cancer Res; 71(13); 4608–16. ©2011 AACR.

Supplementary Video 7 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Methods from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 6 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 5 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 4 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure 3 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Figure Legends 1-4, Movie Legends 1-8 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 8 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Video 3 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Supplementary Table 4 from Analysis of Mitosis and Antimitotic Drug Responses in Tumors by In Vivo Microscopy and Single-Cell Pharmacodynamics

Macrophages often abound within tumors, express colony-stimulating factor 1 receptor (CSF1R), and are linked to adverse patient survival. Drugs blocking CSF1R signaling have been used to suppress tumor-promoting macrophage responses; however, their mechanisms of action remain incompletely understood. Here, we assessed the lung tumor immune microenvironment in mice treated with BLZ945, a prototypical small-molecule CSF1R inhibitor, using single-cell RNA sequencing and mechanistic validation approaches. We showed that tumor control was not caused by CSF1R+ cell depletion; instead, CSF1R targeting reshaped the CSF1R+ cell landscape, which unlocked cross-talk between antitumoral CSF1R− cells. These cells included IFNγ-producing natural killer and T cells, and an IL12-producing dendritic cell subset, denoted as DC3, which were all necessary for CSF1R inhibitor–mediated lung tumor control. These data indicate that CSF1R targeting can activate a cardinal cross-talk between cells that are not macrophages and that are essential to mediate the effects of T cell–targeted immunotherapies and promote antitumor immunity. See related Spotlight by Burrello and de Visser, p. 4 .

Although viridins like wortmannin (Wm) have long been examined as anticancer agents, their ability to self-activate has only recently been recognized. Here, we describe the cytostatic effects of a self-activating viridin (SAV), which is an inactive, polymeric prodrug. SAV self-activates to generate a bioactive, fluorescent viridin NBD-Wm with a half-time of 9.2 hours. With cultured A549 cells, 10 μmol/L SAV caused growth arrest without inducing apoptosis or cell death, a cytostatic action markedly different from other chemotherapeutic agents (vinblastine, camptothecin, and paclitaxel). In vivo, a SAV dosing of 1 mg/kg once in 48 hours (i.p.) resulted in growth arrest of an A549 tumor xenograft, with growth resuming when dosing ceased. With a peak serum concentration of SAV of 2.36 μmol/L (at 2 hours post i.p. injection), the concentration of bioactive NBD-Wm was 41 nmol/L based on the partial inhibition of neutrophil respiratory burst. Therefore, SAV was present as an inactive prodrug in serum (peak = 2.36 μmol/L), which generated low concentrations of active viridin (41 nmol/L). SAV is a prodrug, the slow release and cytostatic activities of which suggest that it might be useful as a component of metronomic-based chemotherapeutic strategies. [Mol Cancer Ther 2009;8(6):1666–75]

Cancer cells often acquire resistance to therapy due to genetic mutations. However, without drug selection, these mutations would likely have a fitness cost to cancer cells and we hypothesize that the severity of this fitness cost is correlated with the effectiveness of drug treatment, with more effective treatments leading to more drastic genetic lesions. The size of a genetic lesion often correlates with its fitness cost - point mutation having little cost, aneuploidy having a large cost. However, aneuploidy is fast because the search space is only 46 gains or losses, while point mutations are slow with billions of base pairs to search before finding the right mutations. We designed experimental and computational systems to understand the selection pressures that would lead to resistance by these different genetic mechanisms. Mice were implanted with xenograft tumors from the HT1080 fibrosarcoma cell line that were heterozygous for DR5 (TNFRSF10B) and extremely sensitive to treatment with a DR5 specific monoclonal antibody, which induced extrinsic apoptosis. Treatment with three doses of anti-DR5 cured 3/11 mice, while 8/11 tumors relapsed. Isolation and culture of the relapse tumors showed they were completely resistant to DR5-mediated killing and had lost the functional copy of DR5. Assaying copy number variation using next generation sequencing (CNVSeq) showed that the relapse lines had lost one copy of chromosome 8p, where the functional DR5 gene was located. Treatment with two doses of anti-DR5 lead to relapses in all mice with loss of 8p. Finally, treatment with a single dose resulted in 3/4 tumors with loss of 8p. However 1/4 tumors became more resistant to anti-DR5 but did not lose the DR5 gene, presumably becoming resistant by smaller genetic changes or by non-genetic means that altered DR5 signaling. Thus, in most cases, treatment of xenograft tumors led to resistance by the loss of a chromosome arm, while resistance was rarely induced by other mechanisms. In contrast, cultured HT1080 cells treated with three doses of anti-DR5 became resistant but never lost 8p. In culture, treatment with anti-DR5 only kills a maximum of 80% of the cells and was therefore weaker than in mice where anti-DR5 could be curative. Thus, we observed an association between the strength of drug selection and the genetic mechanism by which cells became resistant. Experiments to further decrease selective pressure in xenografts and increase it in culture are ongoing. Next, we developed a computational model of tumor evolution to further explore the interplay between strength of selection and type of adaptation. In this simulation, cells were under two selective pressures, drug selection and competition for nutrients. Drug sensitive tumor cells could become resistant by two mechanisms analogous to point mutations or loss of 8p. Point mutations in this model caused resistance incrementally thereby taking several generations to acquire resistance, but they had no fitness cost in terms of competing for nutrients. Loss of 8p in this model caused complete resistance but had a fitness cost. Simulations with strong selective pressure led to the emergence of tumors that had completely lost 8p, even when the fitness cost was high. This result occurred because cells that did not lose 8p were eliminated by the drug. In contrast, simulations with selective pressure similar to culture (80% kill) led to tumors that became resistant without loss of 8p. In this case, sensitive cells incrementally acquired resistance mutations and then out competed cells that lost 8p for nutrients. Thus, simulation showed that resistance mutations with a large fitness cost will only arise under strong drug selection. In conclusion, our data suggest that the closer a therapy is to curing a tumor, the more extreme and costly resistance mutations will be. Citation Format: Lee A. Albacker, Rainer Kohler, Ralph Weissleder, Peter K. Sorger. The strength of drug selection determines the maximum fitness cost of resistance mutations in culture and xenografts. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR07.

Despite immense progress in targeted cancer drug design, to date, the most important drugs in chemotherapy remain traditional cytotoxic drugs which are thought to kill proliferating cells, no matter if malignant of not. This "antiproliferative" hypothesis is based on results from the 1970s coming from mouse leukemia models or cell culture experiments. Microtubule binding drugs are clinically important antiproliferative drugs which arrest cultured cancer cells in mitosis. As opposed to empirically found microtubule binding drugs, newer drugs designed from scratch to induce mitotic arrest have repeatedly failed in clinical trials, suggesting that the antitumor effect of microtubule binding drugs is still not well understood. Alas, tissue culture assays are not predictive of their respective clinical success, suggesting that more realistic models are needed to understand why there are such huge differences in antitumor efficacy. Therefore, in this project, we used in vivo microscopy of mouse xenografts to compare the cell cycle effects of microtubule drugs to those of newer, targeted antimitotic drugs in the form of inhibitors of the mitotic kinesin Eg5. Image analysis with a custom developed software for automated three dimensional segmentation and cell cycle classification revealed that the microtubule interacting drugs Paclitaxel and Eribulin which are in wide clinical use induce a much lower mitotic arrest than the Eg5 inhibitor Ispinesib which failed clinical trials. This suggests that, rather than mitotic arrest, a different mechanism, intrinsically coupled to microtubule binding, might be essential for the antitumor effect of microtubule drugs in vivo. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A297. Citation Format: Stefan Florian, Deepak R. Chittajallu, Rainer H. Kohler, James D. Orth, Peter K. Sorger, Ralph Weissleder, Gaudenz Danuser, Timothy J. Mitchison. Microtubule targeting antimitotic drugs induce a lower mitotic arrest than clinically less effective antimitotic Eg5 inhibitors in mouse xenografts. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A297.

PARP is an enzyme involved in sensing single-stranded DNA breaks and plays a critical role in the organization of DNA damage repair. Several PARP inhibitors have been developed for clinical use, particularly in breast and ovarian cancers with BRCA mutations. Despite the scientific rationale and promising early clinical results with PARP inhibitors, more advanced clinical trials in breast cancer have been disappointing. One of the questions arising from these trials is to better understand why the PARP inhibitors failed. We used a window-chamber system in mice to image the distribution and function of model PARP inhibitors such as AZD2281-Bodipy. Specifically, we measured the kinetics of vascular, cellular, and subcellular uptake and distribution of AZD2281-Bodipy in breast (MDA-MB-231, MDA-MB-436) and ovarian (OVCAR429, A2780) tumors over time. Additionally, using the fluorescently-labeled inhibitor, we were able to examine the heterogeneity of AZD2281 localization in single cancer cells in vivo. Our data provide the first example of single-cell distribution of fluorescent small molecule inhibitors. When coupled with reporter cell lines (e.g. for apoptosis, DNA damage, autophagy, and senescence), this approach will be a powerful tool to understand drug action in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4293. doi:1538-7445.AM2012-4293