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. 2023 Feb 13:12:e82934.
doi: 10.7554/eLife.82934.

Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1

Rui Zhang #  1 , Qingxi Liu #  2   3 , Sa Zhou  4 , Hongpeng He  4 , Mingfeng Zhao  1 , Wenjian Ma  3   4
Affiliations

Mesenchymal stem cell suppresses the efficacy of CAR-T toward killing lymphoma cells by modulating the microenvironment through stanniocalcin-1

Rui Zhang et al. Elife. .

Abstract

Stem cells play critical roles both in the development of cancer and therapy resistance. Although mesenchymal stem cells (MSCs) can actively migrate to tumor sites, their impact on chimeric antigen receptor modified T cell (CAR-T) immunotherapy has been little addressed. Using an in vitro cell co-culture model including lymphoma cells and macrophages, here we report that CAR-T cell-mediated cytotoxicity was significantly inhibited in the presence of MSCs. MSCs caused an increase of CD4+ T cells and Treg cells but a decrease of CD8+ T cells. In addition, MSCs stimulated the expression of indoleamine 2,3-dioxygenase and programmed cell death-ligand 1 which contributes to the immune-suppressive function of tumors. Moreover, MSCs suppressed key components of the NLRP3 inflammasome by modulating mitochondrial reactive oxygen species release. Interestingly, all these suppressive events hindering CAR-T efficacy could be abrogated if the stanniocalcin-1 (STC1) gene, which encodes the glycoprotein hormone STC-1, was knockdown in MSC. Using xenograft mice, we confirmed that CAR-T function could also be inhibited by MSC in vivo, and STC1 played a critical role. These data revealed a novel function of MSC and STC-1 in suppressing CAR-T efficacy, which should be considered in cancer therapy and may also have potential applications in controlling the toxicity arising from the excessive immune response.

Keywords: CAR-T; cancer biology; cancer therapy; human; macrophages; mesenchymal stem cells; pfeiffer cells; regenerative medicine; stanniocalcin-1; stem cells.

Plain language summary

Immunotherapy is a type of cancer treatment that helps the immune system fight cancer. For example, chimeric antigen receptor T cell (CAR-T) therapy is used to target several types of blood cancer. It works by reprogramming patients’ immune cells to target specific tumor cells. In blood cancers, CAR-T therapy works very well, but it can cause extreme responses from the patient’s immune system, which can be life threatening. In solid tumors, CAR-T therapy is much less successful because the tumors secrete molecules into the space surrounding them, which weaken the immune processes that attack cancerous cells. Stem cells are the master cells of the body. Originating in the bone marrow, they can repair and regenerate the body’s cells. Cancer stem cells play a role in resistance to CAR-T therapy, due – in part – to their ability to renew themselves, but the role of another type of stem cell, called mesenchymal stem cells, was less clear. Mesenchymal stem cells develop into tissues that line organs and blood vessels. Although it is known that mesenchymal stem cells are present in most cancers and play a role in shaping and influencing the space around tumors, their impact on CAR-T therapy has not been studied in depth. To find out more, Zhang et al. looked at the influence of a protein, called staniocalcin-1 (STC1), on CAR-T therapy, by studying cells grown in the laboratory and human tumor cells that had been implanted in mice. Zhang et al. found that mesenchymal stem cells reduce the ability of CAR-T therapy to destroy cancer cells and that they needed STC1 to do this successfully. They also increased the expression of molecules that dampen the immune system, and suppressed molecules called inflammasomes, which are an important part of the way the immune system detects disease. Moreover, reducing the amount of STC1 that mesenchymal stem cells expressed restored the effectivity of CAR-T therapy. This study increases our understanding of the way that mesenchymal stem cells affect CAR-T therapy. It has the potential to open up a new way of improving the efficiency of this treatment and of reducing the harmful side effects that it can cause.

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Conflict of interest statement

RZ, QL, SZ, HH, MZ, WM No competing interests declared

Figures

Figure 1.
Figure 1.. The impact of stanniocalcin-1 (STC1) knockdown on cell proliferation, migration, and apoptosis of hMSCs.
(A) Western blot analysis of STC1 protein expression in hMSCs. (B) Cell viability determined by MTT, measurements are shown as the mean ± SD from three independent experiments. (C) FACS analysis of cell cycle progression on hMSCs w/o STC1 knockdown. (D, E) Knockdown of STC1 suppressed cell migration as determined by wound healing and transwell chamber assays. (F) Apoptosis determination by the Alexa Fluor 488 annexin V and PI detection. (G) DNA fragmentation determination by transferase-mediated dUTP nick-end labeling (TUNEL) assay.
Figure 2.
Figure 2.. Analysis of cytotoxicity, T cell composition, and immune-suppressive markers.
The cell co-culture contained chimeric antigen receptor modified T cell (CAR-T) cells, Pfeiffer cells, M2 macrophages, and control or stanniocalcin-1 (STC1) knockdown hMSCs in a ratio of 1:3:1:1. After 24 hr (or 48 hr for cytotoxicity) incubation, the following analysis was conducted: (A) The impact of hMSC (w/o STC1) on the cytotoxicity of CAR-T toward Pfeiffer cells; (B) FACS analysis of CD4+ and CD8+ composition. (C) Quantitation of the FACS data on CD4+ and CD8+; (D) FACS analysis of Treg+ cells (CD4+CD127+CD25+); (E) Quantitation of Treg+ cells. (F) Western blot analysis of indoleamine 2,3-dioxygenase (IDO) and programmed cell death-ligand 1 (PD-L1) expression in the cell co-culture. Data in bar graphs are presented as the mean ± SD from three independent experiments (p values are as indicated, n=3).
Figure 3.
Figure 3.. The impact of mesenchymal stem cells (MSCs) on the expression of key components involved in the formation of NLRP3 inflammasome and mitochondrial reactive oxygen species (ROS).
(A) The protein expression of IL-1β, caspase-1, and AIM2 in cell lysates was analyzed by Western blot. (B) Quantitation of IL-1β secretion in the supernatants by ELISA. (C) FACS analysis of ROS level and mitochondria mass with fluorescent dye CellROX Deep Red and MitoTracker Green. (D) Quantitation of mitochondria-specific ROS level based on the percentage of cells that were both positive for CellROX and MitoTracker. All samples were collected 24 hr post the co-culture of different cells. For the measurements of IL-β, results are shown as the mean ± SD from three independent experiments (p values are as indicated, n=3).
Figure 4.
Figure 4.. The inhibition of hMSC on chimeric antigen receptor modified T cell (CAR-T) therapy in xenograft mice relied on stanniocalcin-1.
(A) The formation and progression of tumor in three groups of mice monitored with bioluminescence imaging: the control group without any treatment, CAR-T/M-THP1/hMSCsshSTC1 group, and CAR-T/M-THP1/hMSCsshCtrl group. Day 0 was set when the engraftment was confirmed after injecting the Pfeiffer cells. (B) Immunohistochemical analysis of IL-1β, CD4+, CD8+, and Treg cells (using FOXP3 as the biomarker) in tumor tissue at day 10, positive cells display brown or brownish-yellow staining color. (C) The tumor size change with time. (D) The counted average radiance, presented as the mean ± SD (p values are as indicated, n=3).
Figure 5.
Figure 5.. Proposed signaling and interactions among hMSC, macrophage, chimeric antigen receptor modified T cell (CAR-T), and tumor cells.
When cancer cells were destroyed by CAR-T cells, the release of fragmented DNA and other stimulating factors activated the release of mitochondria reactive oxygen species (ROS) and the formation of NLPR3 inflammasome. Signals from activated macrophages and other extracellular molecules as well as oxidative stress may stimulate mesenchymal stem cell (MSC) to express and secrete stanniocalcin-1 (STC1). Then STC1 serves as a pleiotropic factor to suppress CAR-T cytotoxicity and other immune responses via direct or indirect pathways.
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Update of

  • doi: 10.1101/2022.09.21.508926

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