Pingwei Xu

Soft fibrin gels promote selection and growth of tumorigenic cells

The identification of stem-cell-like cancer cells through conventional methods that depend on stem-cell markers is often unreliable. We developed a mechanical method of selecting tumourigenic cells by culturing single cancer cells in fibrin matrices of ~100 Pa in stiffness. When cultured within these gels, primary human cancer cells or single cancer cells from mouse or human cancer cell lines grew within a few days into individual round colonies that resembled embryonic stem-cell colonies. Subcutaneous or intravenous injection of 10 or 100 fibrin-cultured cells in syngeneic or severe-combined-immunodeficiency mice led to the formation of solid tumours at the site of injection or at the distant lung organ much more efficiently than control cancer cells selected using conventional surface marker methods or cultured on conventional rigid dishes or on soft gels. Remarkably, as few as 10 such cells were able to survive and form tumours in the lungs of wild-type non-syngeneic mice.

Delivery of chemotherapeutic drugs in tumour cell-derived microparticles

Cellular microparticles are vesicular plasma membrane fragments with a diameter of 100-1,000 nanometres that are shed by cells in response to various physiological and artificial stimuli. Here we demonstrate that tumour cell-derived microparticles can be used as vectors to deliver chemotherapeutic drugs. We show that tumour cells incubated with chemotherapeutic drugs package these drugs into microparticles, which can be collected and used to effectively kill tumour cells in murine tumour models without typical side effects. We describe several mechanisms involved in this process, including uptake of drug-containing microparticles by tumour cells, synthesis of additional drug-packaging microparticles by these cells that contribute to the cytotoxic effect and the inhibition of drug efflux from tumour cells. This study highlights a novel drug delivery strategy with potential clinical application.

Innate Immune Cell–Derived Microparticles Facilitate Hepatocarcinoma Metastasis by Transferring Integrin αMβ2 to Tumor Cells

Mechanisms by which tumor cells metastasize to distant organs still remain enigmatic. Immune cells have been assumed to be the root of metastasis by their fusing with tumor cells. This fusion theory, although interpreting tumor metastasis analogically and intriguingly, is arguable to date. We show in this study an alternative explanation by immune cell–derived microparticles (MPs). Upon stimulation by PMA or tumor cell–derived supernatants, immune cells released membrane-based MPs, which were taken up by H22 tumor cells, leading to tumor cell migration in vitro and metastasis in vivo. The underlying molecular basis was involved in integrin αMβ2 (CD11b/CD18), which could be effectively relayed from stimulated innate immune cells to MPs, then to tumor cells. Blocking either CD11b or CD18 led to significant decreases in MP-mediated tumor cell metastasis. This MP-mediated transfer of immune phenotype to tumor cells might also occur in vivo. These findings suggest that tumor cells may usurp innate immune cell phenotypes via MP pathway for their metastasis, providing new insight into tumor metastatic mechanism.

Spontaneous Miscarriages Are Explained by the Stress/Glucocorticoid/Lipoxin A4 Axis

Despite various suspected causes, ranging from endocrine and genetic to infectious and immunological aspects, the molecular mechanisms of miscarriage still remain enigmatic. This work provides evidence that downregulation of 11β-hydroxysteroid dehydrogenase (HSD) type 2, the key enzyme inactivating glucocorticoid activities, insults the pregnant inflammatory milieu by inhibiting the biosynthesis of lipoxin A4 (LXA4), a metabolite of arachidonic acid, leading to an early loss of the pregnancy. Both LXA4 and its biosynthetic enzymes were found to be decreased in women with spontaneous miscarriages and in the murine miscarriage model. Replenishing LXA4 reversed LPS-induced miscarriages in mouse models, whereas blocking LXA4 signaling resulted in miscarriages in the pregnant mice. The protective effect of LXA4 might be explained by LXA4’s role in regulating uterine and placental inflammatory factors and mast cells. The underlying molecular mechanism involved miscarriage-inducing infections or stresses that downregulate the expression of 11β-HSD2, but not 11β-HSD1, resulting in increases in glucocorticoid activity and decreases in LXA4. Together, these findings suggest that the stress/glucocorticoid/LXA4 axis might be a common pathway through which miscarriages occur.

Upregulation of Cytosolic Phosphoenolpyruvate Carboxykinase Is a Critical Metabolic Event in Melanoma Cells That Repopulate Tumors

Although metabolic defects have been investigated extensively in differentiated tumor cells, much less attention has been directed to the metabolic properties of stem-like cells that repopulate tumors [tumor-repopulating cells (TRC)]. Here, we show that melanoma TRCs cultured in three-dimensional soft fibrin gels reprogram glucose metabolism by hijacking the cytosolic enzyme phosphoenolpyruvate carboxykinase (PCK1), a key player in gluconeogenesis. Surprisingly, upregulated PCK1 in TRCs did not mediate gluconeogenesis but promoted glucose side-branch metabolism, including in the serine and glycerol-3-phosphate pathways. Moreover, this retrograde glucose carbon flow strengthened rather than antagonized glycolysis and glucose consumption. Silencing PCK1 or inhibiting its enzymatic activity slowed the growth of TRCs in vitro and impeded tumorigenesis in vivo. Overall, our work unveiled metabolic features of TRCs in melanoma that have implications for targeting a unique aspect of this disease.

Reversing drug resistance of soft tumor-repopulating cells by tumor cell-derived chemotherapeutic microparticles

Developing novel approaches to reverse the drug resistance of tumor-repopulating cells (TRCs) or stem cell-like cancer cells is an urgent clinical need to improve outcomes of cancer patients. Here we show an innovative approach that reverses drug resistance of TRCs using tumor cell-derived microparticles (T-MPs) containing anti-tumor drugs. TRCs, by virtue of being more deformable than differentiated cancer cells, preferentially take up T-MPs that release anti-tumor drugs after entering cells, which in turn lead to death of TRCs. The underlying mechanisms include interfering with drug efflux and promoting nuclear entry of the drugs. Our findings demonstrate the importance of tumor cell softness in uptake of T-MPs and effectiveness of a novel approach in reversing drug resistance of TRCs with promising clinical applications.

Microparticles released by Listeria monocytogenes -infected macrophages are required for dendritic cell-elicited protective immunity

Interplay between macrophages and dendritic cells in the processing and presentation of bacterial antigens for T-cell immune responses remains poorly understood. Using a Listeria monocytogenes (Lm) infection model, we demonstrate that dendritic cells (DCs) require the support of macrophages to elicit protective immunity against Lm infection. DCs themselves were inefficient at taking up Lm but capable of taking up microparticles (MPs) released by Lm-infected macrophages. These MPs transferred Lm antigens to DCs, allowing DCs to present Lm antigen to effector T cells. MP-mediated Lm antigen transfer required MHC class I participation, since MHC class I deficiency in macrophages resulted in a significant reduction of T-cell activation. Moreover, the vaccination of mice with MPs from Lm-infected macrophages produced strong protective immunity against Lm infection. We here identify an intrinsic antigen transfer program between macrophages and DCs during Lm infection, and emphasize that macrophages also play an essential role in DC-elicited Lm-specific T-cell responses.

Pre-instillation of tumor microparticles enhances intravesical chemotherapy of nonmuscle-invasive bladder cancer through a lysosomal pathway

Nonmuscle-invasive bladder cancer (NMIBC) is treated with transurethral resection followed by intravesical chemotherapy. However, drug-resistant tumorigenic cells cannot be eliminated, leading to half of the treated cancers recur with increased stage and grade. Innovative approaches to enhance drug sensitivity and eradicate tumorigenic cells in NMIBC treatment are urgently needed. Here, we show that pre-instillation of tumor cell-derived microparticles (T-MP) as natural biomaterials markedly enhance the inhibitory effects of intravesical chemotherapy on growth and hematuria occurrence of orthotropic bladder cancer in mice. We provide evidence that T-MPs enter and increase the pH value of lysosomes from 4.6 to 5.6, leading to the migration of drug-loaded lysosomes along microtubule tracks toward the nucleus and discharging the drugs whereby for the entry of the nucleus. We propose that T-MPs may function as a potent sensitizer for augmenting NMIBC chemotherapy with unprecedented clinical benefits.

Downregulation of PCK2 remodels tricarboxylic acid cycle in tumor-repopulating cells of melanoma

For cancer cells to proliferate, a balance must be built between biomass-forming, glucose-metabolized intermediates and ATP production. How intrinsic glucose carbon flow regulates this balance remains unclear. Here we show that mitochondrial phosphoenolpyruvate carboxykinase (PCK2), the hub molecule linking tricarboxylic acid (TCA) cycle, glycolysis and gluconeogenesis by conversion of mitochondrial oxaloacetate (OAA) to phosphoenolpyruvate, regulates glucose carbon flow direction in stem-like cells that repopulate tumors (tumor-repopulating cells (TRCs)). PCK2 downregulation accelerated biosynthesis and transportation of citrate from mitochondria to the cytosol, leading to cytosolic glucose carbon flow via OAA–malate–pyruvate and acetyl-CoA–fatty acid pathways in TRCs. On the other hand, downregulating PCK2 hindered fumarate carbon flows in TCA cycle, leading to attenuated oxidative phosphorylation. In pathological terms, PCK2 overexpression slowed TRC growth in vitro and impeded tumorigenesis in vivo. Overall, our work unveiled unexpected glucose carbon flows of TRCs in melanoma that have implications for targeting metabolic aspects of melanoma.

Mechanisms by Which Dendritic Cells Present Tumor Microparticle Antigens to CD8+ T Cells

Tumor-derived microparticles activate a lysosomal pathway enabling dendritic cell upregulation of costimulatory molecules and presentation of tumor antigens to CD+ T cells. Elucidation of this molecular pathway has clinical implications for the development of improved cancer vaccines. Tumor cell–derived microparticles (T-MP) contain tumor antigen profiles as well as innate signals, endowing them with vaccine potential; however, the precise mechanism by which DCs present T-MP antigens to T cells remains unclear. Here, we show that T-MPs activate a lysosomal pathway that is required for DCs presenting tumor antigens of T-MPs. DCs endocytose T-MPs to lysosomes, where T-MPs increase lysosomal pH from 5.0 to a peak of 8.5 via NOX2-catalyzed reactive oxygen species (ROS) production. This increased pH, coupled with T-MP–driven lysosomal centripetal migration, promotes the formation of MHC class I–tumor antigen peptide complexes. Concurrently, endocytosis of T-MPs results in the upregulation of CD80 and CD86. T-MP–increased ROS activate lysosomal Ca2+ channel Mcoln2, leading to Ca2+ release. Released Ca2+ activates transcription factor EB (TFEB), a lysosomal master regulator that directly binds to CD80 and CD86 promoters, promoting gene expression. These findings elucidate a pathway through which DCs efficiently present tumor antigen from T-MPs to CD8+ T cells, potentiating T-MPs as a novel tumor cell–free vaccine with clinical applications. Cancer Immunol Res; 6(9); 1057–68. ©2018 AACR.