Banovich, Nicholas E.

Abstract Peritoneal Carcinomatosis (PC) is a metastatic cancer of the lining of the abdominal cavity, most often originating from the gastrointestinal or gynecological tracts. PC occurs in approximately 20% of colorectal cancer and 60% of ovarian cancer cases, with estimated 5-year survival rates as low as 6%. Pressurized intraperitoneal aerosolized chemotherapy (PIPAC) is a locoregional drug delivery modality administered via a minimally invasive laparoscopic approach. PIPAC shows promise in the treatment of peritoneal metastasis in early trials on colorectal cancer (CRC) or appendiceal cancer (AC) in patients who cannot undergo cytoreductive surgery. The spatial organization of the tumor microenvironment (TME) and the complex cellular interactions within profoundly shape biology and treatment response. Spatial transcriptomics offers an opportunity to characterize TME heterogeneity in its native architecture, allowing the identification of biomarkers, interactions, and gene expression programs as well as resistance mechanisms and new potential targets. We performed image-based spatial transcriptomic profiling of 152 tissue samples, including 50 pre-treatment and 65 post-treatment metastatic tumors and 17 pre-treatment and 20 post-treatment tumor-adjacent normal tissue samples from 19 patients who participated in a Phase I trial on Oxaliplatin PIPAC. Through a comprehensive characterization of transcriptional profiles of a total of 1,249,711 cells, as well as cellular neighborhood and ligand-receptor interactions, we describe the heterogeneity of PC in its spatial context. We have further characterized treatment-driven changes in pre- and post-treatment tumors of a subset of 7 patients who underwent PIPAC with mitomycin IP. Through cell-based niche and proximity analyses, we identify distinct shifts in the composition and architecture of treated tumors. Most notably, treatment resulted in an 87% decrease in the number of malignant cells, as well as an expansion of the plasma-barrier niche and an increased number of immune infiltrates. Further analysis revealed distinct differences in the abundance of specific cancer-associated fibroblast and immune subsets, demonstrating changes in TME architecture. Overall, our results provide an invaluable characterization of PC in CRC and AC, garnering new insight into their architecture, cellular composition, and cell-cell interactions at baseline and in response to regional chemotherapy. Further analyses and follow-up studies can identify and validate new, actionable biomarkers to target these hard-to-treat tumors. Citation Format: Heini Maaret Natri, Arianna L. Williams-Katek, Muhammad Talha Waheed, Tiana Li, Marwan Fakih, Mingye Feng, Amit Merchea, Sue Chang, Richard L. Whelan, Danielle Deperalta, Thanh H. Dellinger, Nicholas Banovich, Mustafa Raoof. Spatial transcriptomic profiling of peritoneal metastases identifies multicellular programs in patient tumors undergoing pressurized intraperitoneal aerosol chemotherapy (PIPAC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 3958.

Abstract Previously, the IAP antagonist LCL161 was shown to have robust anti-myeloma activity in a transgenic mouse model where an acute inflammatory response accompanied a reduction in tumor burden. In order to fully examine alterations to the immune microenvironment, murine splenocytes from twenty mice transplanted with Vk12598 myeloma and administered single-agent treatment regimens of LCL161, cyclophosphamide, or bortezomib were harvested after 24 hours and subjected to single cell RNA-seq sequencing. After initial removal of erythrocytic and mitochondrial expressing cells, count matrices of over 50,000 total cells span twenty murine whole spleen samples. In an examination of the myeloid component, we identified 13 clusters of immune cells with unique compositions across samples due to differing treatment regimens and initial tumor burden. While minimally present in all seven non-transplanted mice, macrophages with elevated expression of complement activation system genes (C1q) were the most abundant myeloid cell population in each of our untreated, Vk12598 transplanted mice. Alternatively, populations of cDC2, pDC, moDC, and monocytes lacking expression of MHC class II markers were fixtures for healthy controls while markedly less abundant in tumor-bearing mice. Tumor burdens in three samples subjected to LCL161 therapy were significantly reduced 24-hours post treatment and accompanied by an expansion of late-stage neutrophils and migratory DCs with expression features mirroring those of DCs from mesenteric lymph nodes. This composition of immune cells was distinct from all other sample including those treated with cyclophosphamide or bortezomib. In addition, relative proportions of B, T, and NK cells were nearly fully reconstituted, matching levels observed in healthy controls, in two of these samples. In a direct functional assessment, expression levels of Fscn1 and Ccr7 (key over-expressed genes in these migratory DCs) markedly increased following treatment of bone marrow derived DCs with LCL161. Overall, the transcriptional profile of these migratory DCs indicates their pivotal role in maturation of DCs (Fscn1, Tmem123) as well as the recruitment of T and NKT cells (Cxcl16, Ccr7) and increase in antigen presentation (Cd63)–all of which may serve to ‘warm' tumors, increasing T cell infiltration necessary for immunotherapy approaches. With the increased resolution of scRNA-seq, we observe unique alterations in the composition of the immune microenvironment of MM induced by treatment with LCL161 that further detail its unique mechanism of anti-MM activity. Citation Format: Caleb K. Stein, Erin Meermeier, Seth Welsh, Victoria Garbitt, Meaghen Sharik, P. Leif Bergsagel, Nicholas Banovich, Jonathan Keats, Marta Chesi. scRNA-seq reveals restructuring of the immune microenvironment of multiple myeloma following treatment with an IAP antagonist [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5622.

Abstract Multiple myeloma (MM) is the second most common hematologic cancer, accounting for roughly 2% of all cancer deaths. Unfortunately, MM remains incurable and nearly all patients experience relapse. Recently, we have identified a novel cIAP1 antagonist, LCL161, with remarkable anti-MM effects. Interestingly, unlike in other tumor types, our previous work found that LCL161 does not directly induce apoptosis of MM cells, but rather drives activation and infiltration of macrophages to the tumor bed and phagocytosis of tumor cells. Furthermore, in vitro experiments demonstrate that the anti-MM activity of the agent relies on the presence of the tumor, with no observed increase in MM phagocytosis when macrophages are treated in isolation. However, LCL161 is a strong and indiscriminate transcriptional activator and thus, the molecular mechanisms underlying this immune response in MM remain poorly understood. In an effort to characterize molecular signatures associated with this response, we collected whole spleens from seven WT mice and eleven mice with VK12598 transplanted myeloma for single cell RNA-seq. Of the eleven tumor-bearing mice, three remained untreated, three were treated with LCL161, three with bortezomib, and three with cyclophosphamide. Additionally, one of the WT mice was treated with LCL161. Using the 10X genomics chromium platform, we measured mRNA levels in over 90,000 individual cells. We identified groups of cells with shared expression profiles, corresponding to known cell types, using graph-based clustering. Consistent with our previous work, we observed a marked reduction in tumor cells and activation of the noncanonical NFkB pathway in mice treated with LCL161. Turning our attention to cells identified as monocytes, with a particular focus on putative macrophages, we sought to identify molecular signatures associated with LCL161 treatment. Indeed, using a binomial model we identified over 500 upregulated genes in LCL161-treated macrophages. However, our previous data suggested that the efficacy of LCL161 was dependent on the presence of tumor cells. Thus, we refined these results by comparing the expression profile of macrophages treated with LCL161 in WT and tumor-bearing mice. These contrasts identified dozens of genes that were significantly upregulated in macrophages from LCL161 treated, tumor-bearing mice. Of note, we identified a small number of genes (including Spp1, Irg1, and Lipg) that were active only in macrophages from mice treated with LCL161 and bearing a transplanted tumor and unexpressed in all other cells. We believe these genes—and the pathways in which they are active—hold promise for the development of novel targeted therapies to better treat MM. Citation Format: Nicholas Banovich, Martin Boateng, Marta Chesi, Leif Bergsagel, Jonathan Keats. Single-cell RNA sequencing identifies macrophage-specific expression signatures associated with phagocytosis of multiple myeloma after treatment with cIAP antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3135.