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Colorectal cancer (CRC) remains one of the deadliest cancers worldwide due to poor outcomes upon metastatic spread. Given the drastic impact on mortality, a better understanding of the CRC metastatic process could yield significant improvements to overall survival. Here, we investigate the influences of biomechanical forces on early metastatic spread (i.e., tumor cell intravasation events) in an organ-on-chip (OOC) model. Our microfluidic CRC OOC model consists of two channels separated by a porous membrane: a top channel representing normal and cancerous colon epithelial tissue, and a bottom channel consisting of endothelial cells in a tube-like structure representing the vasculature. Vacuum channels flank the sides of the model to apply forces in a cyclic manner to mimic peristalsis-like motions and provide further in vivo resemblance. In our prior work, we demonstrated that peristaltic forces promoted tumor cell invasion within the CRC OOC. Specifically using on-chip confocal imaging, we revealed that cyclic strain leads to an increase in the number of tumor cells capable of intravasating from the epithelial channel into the vascular channel. Additionally, we showed dynamic sensitivity of tumor cell invasion in response to intermittent stretching conditions, leading us to hypothesize mechanical stress-induced phenotypic switching. Therefore, using single-cell RNA sequencing, we investigated gene expression changes of tumor cells in the presence or absence of peristaltic forces and found alterations in stemness, adhesion and metabolism-related genes. Using median fluorescence intensity (MFI) from flow cytometry data, we have revealed differences in stem-like properties (e.g., CD44, CD133, LGR5) of tumor cells with and without the influence of peristaltic forces both prior to and after intravasation. Additionally, effluent-based analysis of the OOC has shown cytokine and metabolism changes due to peristaltic forces. Ongoing work involves using flow cytometry to further quantify changes in adhesion and metabolism as well further validation in patient derived organoids-on-a-chip. Taken together, OOCs provide a physiologically relevant model to study early CRC intravasation events, revealing phenotypic changes in response to mechanical stimuli that influence tumor cell stemness and invasiveness. Citation Format: Curran A. Shah, Carly Strelez, Yonatan Amzaleg, Einar B. Gunnarsson, Jasmine Foo, Min Yu, Shannon M. Mumenthaler. Peristalsis based modulation of colorectal cancer cells leads to changes in stemness and invasiveness in an organ on chip model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1323.

SummaryHypoxia is a common feature of many solid tumors due to aberrant proliferation and angiogenesis and has been associated with tumor progression and metastasis. Most of the well- known hypoxia effects are mediated through hypoxia-inducible factors (HIFs), but the long- lasting effect of hypoxia beyond the immediate HIF regulation remains less understood. Here we show that hypoxia exerts a prolonged effect through HIF-independent mechanisms to promote metastasis. Using breast cancer patient-derived circulating tumor cell (CTC) lines and common breast cancer cell lines, we found that hypoxia downregulates tumor intrinsic type I interferon (IFN) signaling and its downstream antigen presentation (AP) machinery in luminal breast cancer cells. This effect does not depend on the canonical HIFs but is mediated, at least partially, via forkhead box protein O1 (FOXO1). Moreover, hypoxia induced IFN/AP suppression can last longer than the hypoxic exposure, presenting a “hypoxic memory” phenotype. Hypoxic memory of IFN/AP downregulation is established by specific hypoxic priming, and cells with hypoxic memory have an enhanced ability for tumorigenesis and metastasis. The histone deacetylase inhibitor (HDACi) Entinostat can erase the hypoxic memory and improve the immune clearance of tumor cells when combined with checkpoint immunotherapies in a syngeneic breast cancer mouse model. These results point to a novel mechanism for hypoxia facilitated tumor progression, through a long-lasting memory that provides advantages for CTCs during the metastatic cascade.

Estrogens attenuate bone turnover by inhibiting both osteoclasts and osteoblasts, in part through antagonizing Runx2. Apparently conflicting, stimulatory effects in osteoblast lineage cells, however, sway the balance between bone resorption and bone formation in favor of the latter. Consistent with this dualism, 17s-estradiol (E2) both stimulates and inhibits Runx2 in a locus-specific manner, and here we provide evidence for such locus-specific regulation of Runx2 by E2 in vivo. We also demonstrate dual, negative and positive, regulation of Runx2-driven alkaline phosphatase (ALP) activity by increasing E2 concentrations in ST2 osteoblast progenitor cells. We further compared the effects of E2 to those of the Selective Estrogen Receptor Modulators (SERMs) raloxifene (ral) and lasofoxifene (las) and the phytoestrogen puerarin. We found that E2 at the physiological concentrations of 0.1–1 nM, as well as ral and las, but not puerarin, antagonize Runx2-driven ALP activity. At ≥10 nM, E2 and puerarin, but not ral or las, stimulate ALP relative to the activity measured at 0.1–1 nM. Contrasting the difference between E2 and SERMs in ST2 cells, they all shared a similar dose-response profile when inhibiting pre-osteoclast proliferation. That ral and las poorly mimic the locus- and concentration-dependent effects of E2 in mesenchymal progenitor cells may help explain their limited clinical efficacy.

Circulating tumor cells (CTC) can be isolated via a minimally invasive blood draw and are considered a “liquid biopsy” of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small-molecule drug screen to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetyl-L-cysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single-cell expansion. RNA-seq analysis of growing clones and nongrowing clones confirmed the effect by NAC, but also indicates that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with predominant senescent features. Despite the challenge in expanding all CTCs, NAC treatment led to establishment of single CTC clones that have similar tumorigenic features. Implications: Through a small molecule screen and validation study, we found that NAC could improve the success of ex vivo expansion of single CTCs by mitigating the initial stress, with the potential to facilitate the investigation of functional heterogeneity in CTCs.

Circulating tumor cells (CTCs) can be isolated via a minimally invasive blood draw and are considered a “liquid biopsy” of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs, and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small molecule drug screening to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetylcysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single cell expansion. RNA-seq analysis of growing clones and non-growing clones confirmed the effect by NAC, but also indicate that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with heterogeneous quiescent and senescent features. Despite the challenge in expanding all CTCs, NAC treatment lead to establishment of single CTC clones that have similar tumorigenic features, which will facilitate future functional analyses.

Circulating tumor cells (CTCs) shed from solid tumors can serve as a minimally invasive liquid biopsy for monitoring disease progression. Because CTCs are rare and heterogeneous, their biological properties need to be investigated at the single cell level, which requires efficient ways to isolate and analyze live single CTCs. Current methods for CTC isolation and identification are either performed on fixed and stained cells or need multiple procedures to isolate pure live CTCs. Here, we used the AccuCyte-RareCyte system to develop a Protocol for Integrated Capture and Retrieval of Ultra-pure single live CTCs using Negative and positive selection (PIC&RUN). The positive selection module of PIC&RUN identifies CTCs based on detection of cancer surface markers and exclusion of immune markers. Combined with a two-step cell picking protocol to retrieve ultrapure single CTCs, the positive selection module is compatible for downstream single cell transcriptomic analysis. The negative selection module of PIC&RUN identifies CTCs based on a live cell dye and the absence of immune markers, allowing retrieval of viable CTCs that are suitable for ex vivo culture. This new assay combines the CTC capture and retrieval in one integrated platform, providing a valuable tool for downstream live CTC analyses.

3041 Background: CTCs are a promising diagnostic and prognostic biomarker in colorectal cancer. The analysis of CTCs at the single-cell level may allow to capture tumor heterogeneity and identify novel prognostic and predictive markers in mCRC. Our aim was to evaluate whether the transcriptome profile of CTCs was associated with progression-free survival (PFS) in pts receiving treatment for mCRC and whether we could identify significant differences based on pts’ ethnicity. Methods: Single CTCs were prospectively collected from 25 pts undergoing treatment for mCRC at the USC/Norris Comprehensive Cancer Center between April and October 2019. Pre-treatment peripheral blood was processed using CTC-FIND comprised filter separation and immunomagnetic depletion of CD45/50-expressing cells to collect ultra-pure CTCs samples. After isolation to single cell, CTCs were processed by single cell RNA-sequence (scRNAseq). Principal component analysis was used for clustering expression data. DESeq2 was used to identify differentially expressed genes between pts with short ( < 150 days) and long term ( > 150 days) PFS, as well as between Hispanic and non-Hispanic ethnicity with control for FDR ( Q < 0.1). Ingenuity pathway analyses (IPA) of enriched pathway networks were performed. Results: Main pts characteristics were as follow: median age = 52 years; M/F = 18/7; Hispanics/non-Hispanics = 7/18; treatment line (n pts) = 1st (5), 2nd (11), ≥ 3rd (9). We identified 59 single CTC and CTC cluster from 22/25 pts (range 1-9, median = 2/pt). scRNAseq analysis identified two separate clusters of pts based on PFS (short term vs long term). Hispanic pts were mainly distributed within the short term PFS cluster. The IPA of the network of top 40 enriched pathways showed several pathways related with metabolism, such as Sirtuin signaling, mitochondrial dysfunction and oxidative phosphorylation, and IL6/JAK/STAT signaling pathways in pts with short term PFS ( Q < 0.05). When comparing Hispanic vs non-Hispanic pts, we detected enrichment of neuroinflammation signaling pathway in Hispanics, including CXCL8 (fold change, FC: 11.04), GAD2 (FC: 4.77), IRAK3 (FC: 17.06), PLCG2 (FC: 4.65) and TYROBP (FC: 8.43) (all Q < 0.05). Conclusions: In our study CTCs transcriptome profiles showed an association with PFS in pts receiving treatment for mCRC, with an enrichment in mitochondria-related pathways and IL6/JAK/STAT signaling in the CTCs of pts with shorter PFS. Additionally, CTCs scRNAseq identified differentially expressed genes in Hispanic pts, displaying enrichment in neuroinflammation signaling. These results highlight the potential of CTCs molecular profiling as a tool to identify novel prognostic and predictive biomarkers in mCRC and actionable molecular pathways which may impact tumor spread and treatment response

An amendment to this paper has been published and can be accessed via a link at the top of the paper.