Cell metabolism-based optimization strategy of CAR-T cell function in cancer therapy

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR)-modified T cells has revolutionized the field of immune-oncology, showing remarkable efficacy against hematological malignancies. However, its success in solid tumors is limited by factors such as easy recurrence and poor efficacy. The effector function and persistence of CAR-T cells are critical to the success of therapy and are modulated by metabolic and nutrient-sensing mechanisms. Moreover, the immunosuppressive tumor microenvironment (TME), characterized by acidity, hypoxia, nutrient depletion, and metabolite accumulation caused by the high metabolic demands of tumor cells, can lead to T cell “exhaustion” and compromise the efficacy of CAR-T cells. In this review, we outline the metabolic characteristics of T cells at different stages of differentiation and summarize how these metabolic programs may be disrupted in the TME. We also discuss potential metabolic approaches to improve the efficacy and persistence of CAR-T cells, providing a new strategy for the clinical application of CAR-T cell therapy

MicroRNA-34c enhances murine male germ cell apoptosis through targeting ATF1.

BACKGROUND: MicroRNAs (miRNAs) play vital regulatory roles in many cellular processes. The expression of miRNA (miR)-34c is highly enriched in adult mouse testis, but its roles and underlying mechanisms of action are not well understood. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we show that miR-34c is detected in mouse pachytene spermatocytes and continues to be highly expressed in spermatids. To explore the specific functions of miR-34c, we have established an in vivo model by transfecting miR-34c inhibitors into primary spermatocytes to study the loss-of-function of miR-34c. The results show that silencing of miR-34c significantly increases the Bcl-2/Bax ratio and prevents germ cell from apoptosis induced by deprivation of testosterone. Moreover, ectopic expression of the miR-34c in GC-2 cell trigger the cell apoptosis with a decreased Bcl-2/Bax ratio and miR-34c inhibition lead to a low spontaneous apoptotic ratio and an increased Bcl-2/Bax ratio. Furthermore, ectopic expression of miR-34c reduces ATF1 protein expression without affecting ATF1 mRNA level via directly binding to ATF1's 3'UTR, indicating that ATF1 is one of miR-34c's target genes. Meanwhile, the knockdown of ATF1 significantly decreases the Bcl-2/Bax ratio and triggers GC-2 cell apoptosis. Inhibition of miR-34c does not decrease the GC-2 cell apoptosis ratio in ATF1 knockdown cells. CONCLUSIONS/SIGNIFICANCE: Our study shows for the first time that miR-34c functions, at least partially, by targeting the ATF1 gene in germ cell apoptosis, providing a novel mechanism with involvement of miRNA in the regulation of germ cell apoptosis

MiR-34c knock-down enhances resistance to cell apoptosis <i>in vivo</i>.

<p>(A) <i>In situ</i> DNA TUNEL labeling after flutamide induction in different treatment groups. Non-transfected: no injection testis; in-NC: inhibitor NC transfection with Lipofectamine 2000 testis as a negative control; inhibitor: miR-34c inhibitor transfection with Lipofectamine 2000 testis. (B) Number of apoptotic germ cells per 100 seminiferous tubules in different treatment group. At least 500 seminiferous tubules were counted for every testis. Each bar presents the mean ± S.E.M of 6 testes from different mice. (*<i>P</i><0.05, **<i>P</i><0.01).</p

The effects of miR-34c inhibition on Fas, Bcl-2, Bax mRNA expressions and Bcl-2/Bax ratio.

<p>(A) MiRNA inhibitor with Lipofectamine™ 2000 injected into the seminiferous tubule of 14 dpp mouse testis. 0.4% Trypan blue (10 fold more than needed for experimental concentrations and used in order to take a clear picture) was added to transfection mix. (B) Real-time PCR analysis two days after injection. (lipo: lipofectamine™; inhibitor: miR-34c inhibitor; in-NC: miRNA inhibitor nonsense control. (C) Apoptosis-related genes were tested by real-time PCR after miR-34c knockdown. (D) Bcl-2/Bax ratio increased after miR-34c inhibition. Each bar presents the mean ± S.E.M of 6 testes from different mice. (*<i>P</i><0.05, **<i>P</i><0.01).</p

MiR-34c binds to ATF1-3′UTR which regulates its protein expression.

<p>(A) The predicted miR-34c binding site in ATF1-3′UTR from <a href="http://www.miRNA.org" target="_blank">www.miRNA.org</a>. (B) Schematic of inserted ATF1-3′UTRs sequences. (C) psi-CHECKTM-2 reporter vector map. (D) Relative luminescence intensity detected by Modulus™II microplate multimode reader after miR-34c mimics and dual-luciferase vector were co-transfected into 293T cells. (E) ATF1 mRNA and protein levels were analyzed by real-time PCR and Western blot after miR-34c inhibition <i>in vivo</i>. (F) ATF1 mRNA and protein levels <i>in vitro</i> in a GC-2 cell culture after miR-34c overexpression or inhibition. Each bar presents the mean ± S.E.M from three samples for each group. (*<i>P</i><0.05, **<i>P</i><0.01).</p

Effects of miR-34c on GC-2 cell apoptosis.

<p>(A) Overexpression and inhibition efficiency of miR-34c mimics or inhibitor, transfected into GC-2 cells after 24 h. Non-treated: no transfection control; mi-NC or in-NC: mimics NC or inhibitor NC transfection with Lipofectamine 2000 as a strict control; mimics 1: 20 nM mimics as a final transfection concentration; mimics 2: 30 nM as a final transfection concentration; inhibitor 1 and inhibitor 2: similar to mimics, 20 nM and 30 nM inhibitor were transfected respectively. (B) Apoptosis-related genes expression after mimics or inhibitor transfection. NC, mi-NC or in-NC as mimics or inhibitor control; Mimics L or inhibitor L: 20 nM mimics or inhibitors were transfected into GC-2 cells. (C) The bcl-2/bax ratio changes when miR-34c is overexpressed or inhibited. (D) GC-2 cell apoptosis analysis by Annexin V/Propidium Iodide staining. (E) The summary of GC-2 cell apoptotic ratio after miR-34c overexpression or inhibition. Each bar presents the mean ± S.E.M from three samples for each group. (**<i>P</i><0.01, ***<i>P</i><0.001).</p

MiR-34c expression is dynamic in immature and adult mouse testes.

<p>(A) Real-time PCR for miR-34c. (B) Localization of miR-34c in mouse testis at different development stages using LNA <i>in situ</i> hybridization. The first five pictures are 12 dpp-adult ISH of miR-34c and a scrambled probe control (sc-adult). The last picture is a PI staining (red) of cell nuclei of the adult section. All the panels are shown at the same magnifications. L - Leptotene spermatocytes; P - Pachytene spermatocytes; Sd - Spermatids. Data were shown as mean ± S.E.M of three samples for each group.</p