Suppression of osteosarcoma progression by engineered lymphocyte-derived proteomes.

Cancer cells tend to develop resistance to chemotherapy and enhance aggressiveness. A counterintuitive approach is to tame aggressiveness by an agent that acts opposite to chemotherapeutic agents. Based on this strategy, induced tumor-suppressing cells (iTSCs) have been generated from tumor cells and mesenchymal stem cells. Here, we examined the possibility of generating iTSCs from lymphocytes by activating PKA signaling for suppressing the progression of osteosarcoma (OS). While lymphocyte-derived CM did not present anti-tumor capabilities, the activation of PKA converted them into iTSCs. Inhibiting PKA conversely generated tumor-promotive secretomes. In a mouse model, PKA-activated CM suppressed tumor-induced bone destruction. Proteomics analysis revealed that moesin (MSN) and calreticulin (Calr), which are highly expressed intracellular proteins in many cancers, were enriched in PKA-activated CM, and they acted as extracellular tumor suppressors through CD44, CD47, and CD91. The study presented a unique option for cancer treatment by generating iTSCs that secret tumor-suppressive proteins such as MSN and Calr. We envision that identifying these tumor suppressors and predicting their binding partners such as CD44, which is an FDA-approved oncogenic target to be inhibited, may contribute to developing targeted protein therapy.

Proteomes from AMPK-inhibited peripheral blood mononuclear cells suppress the progression of breast cancer and bone metastasis.

Background: During a developmental process, embryos employ varying tactics to remove unwanted cells. Using a procedure analogous to some of the embryonic cells, we generated a tumor-eliminating conditioned medium (CM) from AMPK-inhibited lymphocytes and monocytes in peripheral blood mononuclear cells (PBMCs). Methods: AMPK signaling was inhibited by the application of a pharmacological agent, Dorsomorphin, and the therapeutic effects of their conditioned medium (CM) were evaluated using in vitro cell cultures, ex vivo breast cancer tissues, and a mouse model of mammary tumors and tumor-induced osteolysis. The regulatory mechanism was evaluated using mass spectrometry-based proteomics, Western blotting, immunoprecipitation, gene overexpression, and RNA interference. Results: While AMPK signaling acted mostly anti-tumorigenic, we paradoxically inhibited it to build induced tumor-suppressing cells and their tumor-eliminating CM. In a mouse model of breast cancer, the application of AMPK-inhibited lymphocyte-derived CM reduced mammary tumors additively to a chemotherapeutic agent, Taxol. It also prevented bone loss in the tumor-bearing tibia. Furthermore, the application of CM from the patient-derived peripheral blood diminished ex vivo breast cancer tissues isolated from the same patients. Notably, proteins enriched in CM included Moesin (MSN), Enolase 1 (ENO1), and polyA-binding protein 1 (PABPC1), which are considered tumorigenic in many types of cancer. The tumor-suppressing actions of MSN and ENO1 were at least in part mediated by Metadherin (Mtdh), which is known to promote metastatic seeding. Conclusion: We demonstrated that PBMCs can be used to generate tumor-suppressive proteomes, and extracellular tumor-suppressing proteins such as MSN, ENO1, and PABPC1 are converted from tumor-promoting factors inside cancer cells. The results support the possibility of developing autologous blood-based therapy, in which tumor-suppressing proteins are enriched in engineered PBMC-derived CM by the inhibition of AMPK signaling.

Viscoelastic hydrogels for interrogating pancreatic cancer-stromal cell interactions.

The tumor microenvironment (TME) is known to direct cancer cell growth, migration, invasion into the matrix and distant tissues, and to confer drug resistance in cancer cells. While multiple aspects of TME have been studied using in vitro, ex vivo, and in vivo tumor models and engineering tools, the influence of matrix viscoelasticity on pancreatic cancer cells and its associated TME remained largely unexplored. In this contribution, we synthesized a new biomimetic hydrogel with tunable matrix stiffness and stress-relaxation for evaluating the effect of matrix viscoelasticity on pancreatic cancer cell (PCC) behaviors in vitro. Using three simple monomers and Reverse-Addition Fragmentation Chain-Transfer (RAFT) polymerization, we synthesized a new class of phenylboronic acid containing polymers (e.g., poly (OEGA-s-HEAA-s-APBA) or PEHA). Norbornene group was conjugated to HEAA on PEHA via carbic anhydride, affording a new NB and BA dually modified polymer - PEHNBA amenable for orthogonal thiol-norbornene photopolymerization and boronate ester diol complexation. The former provided tunable matrix elasticity, while the latter gave rise to matrix stress-relaxation (or viscoelasticity). The new PEHNBA polymers were shown to be highly cytocompatible for in situ encapsulation of PCCs and cancer-associated fibroblasts (CAFs). Furthermore, we demonstrated that hydrogels with high stress-relaxation promoted spreading of CAFs, which in turns promoted PCC proliferation and spreading in the viscoelastic matrix. Compared with elastic matrix, viscoelastic gels upregulated the secretion of soluble proteins known to promote epithelial-mesenchymal transition (EMT). This study demonstrated the crucial influence of matrix viscoelasticity on pancreatic cancer cell fate and provided an engineered viscoelastic matrix for future studies and applications related to TME.

Ah-type Baroreceptor Neurons Expressing Estrogen Dependent mGluR7 Mediate Descending Inhibition of Cardiac Nociception.

Silent myocardial infarction (MI) is critical for clinical practice with increasing risk for women and the cause remains a medical mystery. Upon the discovery of female-specific Ah-type baroreceptor neurons (BRNs), we hypothesize that glutamate mediates depressor response through afferent-specific expression of particular glutamate receptors (mGluRs) leading descending inhibition of cardiac nociception. In vivo, tail-flick reflex and electromyography were assessed to evaluate glutamate-mediated blood pressure regulation, peripheral and cardiac nociception. The results showed that glutamate decreased mean arterial pressure (MAP) and increased peripheral nociception. Interestingly, glutamate-mediated capsaicin-induced cardiac nociception was strongly reduced in female rats compared with males. Furthermore, Nodose (NG) microinjection of mGluR7 agonist significantly increased MAP in males and slightly decreased that in females. Even though mGluR8 direct activation intensified baroreceptor activation, the sensitivity was similar between sexes. In vitro, the expression profiles of mGluRs were investigated using Western blot and identified BRNs using single-cell qRT-PCR under ischemic conditions. Glutamate in serum, NG and nucleus tractus solitary (NTS) was raised significantly in the model rats of both sexes vs. sham-controls. Female-specific expression of mGluR7 in the baroreflex afferent pathway, especially higher expression in Ah-type BRNs, contributes significantly to cardiac analgesia, which may explain that the pathogenesis of silent MI occurs mainly in female patients. Therefore, higher expression of mGluR7 in female-specific subpopulation of Ah-type BRNs plays a critical role in cardiac analgesia and peripheral nociception.

Estrogen-dependent depressor response of melatonin via baroreflex afferent function and intensification of PKC-mediated Nav1.9 activation.

Recent studies suggest that melatonin (Mel) plays an important role in the regulation of blood pressure (BP) via the aortic baroreflex pathway. In this study, we investigated the interaction between the baroreflex afferent pathway and Mel-mediated BP regulation in rats under physiological and hypertensive conditions. Mel (0.1, 0.3, and 1.0 mg/mL) was microinjected into the nodose ganglia (NG) of rats. We showed that Mel-induced reduction of mean arterial pressure in female rats was significantly greater than that in male and in ovariectomized rats under physiological condition. Consistently, the expression of Mel receptors (MTNRs) in the NG of female rats was significantly higher than that of males. In L-NAME-induced hypertensive and spontaneously hypertensive rat models, MTNRs were upregulated in males but downregulated in female models. Interestingly, Mel-induced BP reduction was found in male hypertensive models. In whole-cell recording from identified baroreceptor neurons (BRNs) in female rats, we found that Mel (0.1 µM) significantly increased the excitability of a female-specific subpopulation of Ah-type BRNs by increasing the Nav1.9 current density via a PKC-mediated pathway. Similar results were observed in baroreceptive neurons of the nucleus tractus solitarius, showing the facilitation of spontaneous and evoked excitatory post-synaptic currents in Ah-type neurons. Collectively, this study reveals the estrogen-dependent effect of Mel/MTNRs under physiological and hypertensive conditions is mainly mediated by Ah-type BRNs, which may provide new theoretical basis and strategies for the gender-specific anti-hypertensive treatment in clinical practice.

Bradykinin-mediated estrogen-dependent depressor response by direct activation of female-specific distribution of myelinated Ah-type baroreceptor neurons in rats.

AIM: To understand the direct impact of bradykinin in autonomic control of circulation through baroreflex afferent pathway. METHODS: The mean arterial pressure (MAP) was monitored while bradykinin and its agonists were applied via nodose (NG) microinjection, the expression of bradykinin receptors (BRs) in the NG (1st -order) and nucleus tractus solitarius (NTS, 2nd -order) were tested in adult male, age-matched female, and ovariectomized rats under physiological and hypertensive conditions. Additionally, bradykinin-induced depolarization was also tested in identified baroreceptor and baroreceptive neurons using whole-cell patch-clamp technique. RESULTS: Under physiological condition, bradykinin-induced dose- and estrogen-dependent reductions of MAP with lower estimated EC50 in females. B2 R agonist mediated more dramatic MAP reduction with long-lasting effect compared with B1 R activation. These functional observations were consistent with the molecular and immunostaining evidences. However, under hypertensive condition, the MAP reduction was significantly less dramatic in N' -Nitro-L-Arginine-methyl ester (L-NAME) induced secondary and spontaneous hypertension rats in males compared with female rats. Electrophysiological data showed that bradykinin-elicited concentration-dependent membrane depolarization with discharges during initial phase in identified myelinated Ah-types baroreceptor neurons, not myelinated A-types; while, higher concentration of bradykinin was required for depolarization of unmyelinated C-types without initial discharges. CONCLUSION: These datasets have demonstrated for the first time that bradykinin mediates direct activation of baroreflex afferent function to trigger estrogen-dependent depressor response, which is due mainly to the direct activation/neuroexcitation of female-specific myelinated Ah-type baroreceptor neurons leading to a sexual dimorphism in parasympathetic domination of blood pressure regulation via activation of B2 R/B1 R expression in baroreflex afferent pathway.

Estrogen-dependent KCa1.1 modulation is essential for retaining neuroexcitation of female-specific subpopulation of myelinated Ah-type baroreceptor neurons in rats.

Female-specific subpopulation of myelinated Ah-type baroreceptor neurons (BRNs) in nodose ganglia is the neuroanatomical base of sexual-dimorphic autonomic control of blood pressure regulation, and KCa1.1 is a key player in modulating the neuroexcitation in nodose ganglia. In this study we investigated the exact mechanisms underlying KCa1.1-mediated neuroexcitation of myelinated Ah-type BRNs in the presence or absence of estrogen. BRNs were isolated from adult ovary intact (OVI) or ovariectomized (OVX) female rats, and identified electrophysiologically and fluorescently. Action potential (AP) and potassium currents were recorded using whole-cell recording. Consistently, myelinated Ah-type BRNs displayed a characteristic discharge pattern and significantly reduced excitability after OVX with narrowed AP duration and faster repolarization largely due to an upregulated iberiotoxin (IbTX)-sensitive component; the changes in AP waveform and repetitive discharge of Ah-types from OVX female rats were reversed by G1 (a selective agonist for estrogen membrane receptor GPR30, 100 nM) and/or IbTX (100 nM). In addition, the effect of G1 on repetitive discharge could be completely blocked by G15 (a selective antagonist for estrogen membrane receptor GPR30, 3 µM). These data suggest that estrogen deficiency by removing ovaries upregulates KCa1.1 channel protein in Ah-type BRNs, and subsequently increases AP repolarization and blunts neuroexcitation through estrogen membrane receptor signaling. Intriguingly, this upregulated KCa1.1 predicted electrophysiologically was confirmed by increased mean fluorescent intensity that was abolished by estrogen treatment. These electrophysiological findings combined with immunostaining and pharmacological manipulations reveal the crucial role of KCa1.1 in modulation of neuroexcitation especially in female-specific subpopulation of myelinated Ah-type BRNs and extend our current understanding of sexual dimorphism of neurocontrol of BP regulation.