Preclinical tumor-immune modeling : For the identification of immunomodulatory drugs

For a long time, the field of cancer research was dominated by a tumor cell-centric view. That, however, changed once it became recognized that medical cancer treatment is largely influenced by the combined effect exerted on both cancer and immune cells. In this work, we aimed to develop and apply preclinical model systems for the identification and evaluation of immunomodulatory anti-cancer agents. In Paper I, we employed single-cell RNA sequencing (scRNA-seq) to investigate immunological effects of trifluridine (FTD), a nucleoside analogue used for the treatment of colorectal cancer (CRC). The study revealed that while FTD induces immunogenic cell death (ICD), it may also attenuate T cell-mediated antitumor responses. In paper II and III, we developed and applied a phenotypic screening platform based on a miniaturized tumor-immune model. In paper II, aiming to identify immunological effects of clinical relevance and provide a reference point for screening novel compound libraries, the model system was used to assess a broad panel of standard anticancer agents. In paper III, the platform was used to screen a drug library containing 1280 small molecule drugs, all approved by the FDA or other agencies. Using this approach, statins were identified as enhancers of immune cell-mediated cancer cell killing. Finally, in paper IV, we developed the immuno-oncology hollow fiber assay (HFA) with the goal of bridging the gap between cell based in vitro assays and more complex mouse models for evaluation of immuno-oncological agents. The HFA is an in vivo assay in which semipermeable fibers are filled with cancer cells and implanted in rodents. We further developed the HFA to incorporate both cancer and immune cells. This novel assay demonstrated the potential to capture immune-mediated cancer cell killing in vivo within a matter of days. Collectively, this work provides a research approach for immuno-oncology drug screening, in vitro validation, and initial in vivo evaluation.

Preclinical tumor-immune modeling : For the identification of immunomodulatory drugs

For a long time, the field of cancer research was dominated by a tumor cell-centric view. That, however, changed once it became recognized that medical cancer treatment is largely influenced by the combined effect exerted on both cancer and immune cells. In this work, we aimed to develop and apply preclinical model systems for the identification and evaluation of immunomodulatory anti-cancer agents. In Paper I, we employed single-cell RNA sequencing (scRNA-seq) to investigate immunological effects of trifluridine (FTD), a nucleoside analogue used for the treatment of colorectal cancer (CRC). The study revealed that while FTD induces immunogenic cell death (ICD), it may also attenuate T cell-mediated antitumor responses. In paper II and III, we developed and applied a phenotypic screening platform based on a miniaturized tumor-immune model. In paper II, aiming to identify immunological effects of clinical relevance and provide a reference point for screening novel compound libraries, the model system was used to assess a broad panel of standard anticancer agents. In paper III, the platform was used to screen a drug library containing 1280 small molecule drugs, all approved by the FDA or other agencies. Using this approach, statins were identified as enhancers of immune cell-mediated cancer cell killing. Finally, in paper IV, we developed the immuno-oncology hollow fiber assay (HFA) with the goal of bridging the gap between cell based in vitro assays and more complex mouse models for evaluation of immuno-oncological agents. The HFA is an in vivo assay in which semipermeable fibers are filled with cancer cells and implanted in rodents. We further developed the HFA to incorporate both cancer and immune cells. This novel assay demonstrated the potential to capture immune-mediated cancer cell killing in vivo within a matter of days. Collectively, this work provides a research approach for immuno-oncology drug screening, in vitro validation, and initial in vivo evaluation.

Single-cell transcriptional pharmacodynamics of trifluridine in a tumor-immune model

Understanding the immunological effects of chemotherapy is of great importance, especially now that we have entered an era where ever-increasing pre-clinical and clinical efforts are put into combining chemotherapy and immunotherapy to combat cancer. Single-cell RNA sequencing (scRNA-seq) has proved to be a powerful technique with a broad range of applications, studies evaluating drug effects in co-cultures of tumor and immune cells are however scarce. We treated a co-culture comprised of human colorectal cancer (CRC) cells and peripheral blood mononuclear cells (PBMCs) with the nucleoside analogue trifluridine (FTD) and used scRNA-seq to analyze posttreatment gene expression profiles in thousands of individual cancer and immune cells concurrently. ScRNA-seq recapitulated major mechanisms of action previously described for FTD and provided new insight into possible treatment-induced effects on T-cell mediated antitumor responses

Phenotypic screening platform identifies statins as enhancers of immune cell-induced cancer cell death

Background: High-throughput screening (HTS) of small molecule drug libraries has greatly facilitated the discovery of new cancer drugs. However, most phenotypic screening platforms used in the field of oncology are based solely on cancer cell populations and do not allow for the identification of immunomodulatory agents. Methods: We developed a phenotypic screening platform based on a miniaturized co-culture system with human colorectal cancer- and immune cells, providing a model that recapitulates part of the tumor immune microenvironment (TIME) complexity while simultaneously being compatible with a simple image-based readout. Using this platform, we screened 1,280 small molecule drugs, all approved by the Food and Drug Administration (FDA), and identified statins as enhancers of immune cell-induced cancer cell death. Results: The lipophilic statin pitavastatin had the most potent anti-cancer effect. Further analysis demonstrated that pitavastatin treatment induced a pro-inflammatory cytokine profile as well as an overall pro-inflammatory gene expression profile in our tumor-immune model. Conclusion: Our study provides an in vitro phenotypic screening approach for the identification of immunomodulatory agents and thus addresses a critical gap in the field of immuno-oncology. Our pilot screen identified statins, a drug family gaining increasing interest as repurposing candidates for cancer treatment, as enhancers of immune cell-induced cancer cell death. We speculate that the clinical benefits described for cancer patients receiving statins are not simply caused by a direct effect on the cancer cells but rather are dependent on the combined effect exerted on both cancer and immune cells

Sorafenib and nitazoxanide disrupt mitochondrial function and inhibit regrowth capacity in three-dimensional models of hepatocellular and colorectal carcinoma

Quiescent cancer cells in malignant tumors can withstand cell-cycle active treatment and cause cancer spread and recurrence. Three-dimensional (3D) cancer cell models have led to the identification of oxidative phosphorylation (OXPHOS) as a context-dependent vulnerability. The limited treatment options for advanced hepatocellular carcinoma (HCC) and colorectal carcinoma (CRC) metastatic to the liver include the multikinase inhibitors sorafenib and regorafenib. Off-target effects of sorafenib and regorafenib are related to OXPHOS inhibition; however the importance of this feature to the effect on tumor cells has not been investigated in 3D models. We began by assessing global transcriptional responses in monolayer cell cultures, then moved on to multicellular tumor spheroids (MCTS) and tumoroids generated from a CRC patient. Cells were treated with chemotherapeutics, kinase inhibitors, and the OXPHOS inhibitors. Cells grown in 3D cultures were sensitive to the OXPHOS inhibitor nitazoxanide, sorafenib, and regorafenib and resistant to other multikinase inhibitors and chemotherapeutic drugs. Furthermore, nitazoxanide and sorafenib reduced viability, regrowth potential and inhibited mitochondrial membrane potential in an additive manner at clinically relevant concentrations. This study demonstrates that the OXPHOS inhibition caused by sorafenib and regorafenib parallels 3D activity and can be further investigated for new combination strategies

Mebendazole is unique among tubulin-active drugs in activating the MEK-ERK pathway

We recently showed that the anti-helminthic compound mebendazole (MBZ) has immunomodulating activity in monocyte/macrophage models and induces ERK signalling. In the present study we investigated whether MBZ induced ERK activation is shared by other tubulin binding agents (TBAs) and if it is observable also in other human cell types. Curated gene signatures for a panel of TBAs in the LINCS Connectivity Map (CMap) database showed a unique strong negative correlation of MBZ with MEK/ERK inhibitors indicating ERK activation also in non-haematological cell lines. L1000 gene expression signatures for MBZ treated THP-1 monocytes also connected negatively to MEK inhibitors. MEK/ERK phosphoprotein activity testing of a number of TBAs showed that only MBZ increased the activity in both THP-1 monocytes and PMA differentiated macrophages. Distal effects on ERK phosphorylation of the substrate P90RSK and release of IL1B followed the same pattern. The effect of MBZ on MEK/ERK phosphorylation was inhibited by RAF/MEK/ERK inhibitors in THP-1 models, CD3/IL2 stimulated PBMCs and a MAPK reporter HEK-293 cell line. MBZ was also shown to increase ERK activity in CD4+ T-cells from lupus patients with known defective ERK signalling. Given these mechanistic features MBZ is suggested suitable for treatment of diseases characterized by defective ERK signalling, notably difficult to treat autoimmune diseases