A fundamental problem in cancer drug development is that antitumor efficacy in preclinical cancer models does not translate faithfully to patient outcomes. Much of early cancer drug discovery is performed under in vitro conditions in cell-based models that poorly represent actual malignancies. To address this inconsistency, we have developed a technology platform called CIVO, which enables simultaneous assessment of up to eight drugs or drug combinations within a single solid tumor in vivo. The platform is currently designed for use in animal models of cancer and patients with superficial tumors but can be modified for investigation of deeper-seated malignancies. In xenograft lymphoma models, CIVO microinjection of well-characterized anticancer agents (vincristine, doxorubicin, mafosfamide, and prednisolone) induced spatially defined cellular changes around sites of drug exposure, specific to the known mechanisms of action of each drug. The observed localized responses predicted responses to systemically delivered drugs in animals. In pair-matched lymphoma models, CIVO correctly demonstrated tumor resistance to doxorubicin and vincristine and an unexpected enhanced sensitivity to mafosfamide in multidrug-resistant lymphomas compared with chemotherapy-naïve lymphomas. A CIVO-enabled in vivo screen of 97 approved oncology agents revealed a novel mTOR (mammalian target of rapamycin) pathway inhibitor that exhibits significantly increased tumor-killing activity in the drug-resistant setting compared with chemotherapy-naïve tumors. Finally, feasibility studies to assess the use of CIVO in human and canine patients demonstrated that microinjection of drugs is toxicity-sparing while inducing robust, easily tracked, drug-specific responses in autochthonous tumors, setting the stage for further application of this technology in clinical trials.

Thin-filament regulation of isometric force redevelopment (k tr ) was examined in rabbit psoas fibres by substituting native TnC with either cardiac TnC (cTnC), a site I-inactive skeletal TnC mutant (xsTnC), or mixtures of native purified skeletal TnC (sTnC) and a site I-and II-inactive skeletal TnC mutant (xxsTnC). Reconstituted maximal Ca 2+ -activated force (rF max ) decreased as the fraction of sTnC in sTnC : xxsTnC mixtures was reduced, but maximal k tr was unaffected until rF max was <0.2 of pre-extracted F max . In contrast, reconstitution with cTnC or xsTnC reduced maximal k tr to 0.48 and 0.44 of control (P < 0.01), respectively, with corresponding rF max of 0.68 ± 0.03 and 0.25 ± 0.02 F max . The k tr -pCa relation of fibres containing sTnC : xxsTnC mixtures (rF max > 0.2 F max ) was little effected, though k tr was slightly elevated at low Ca

We studied the relative contributions of Ca 2+ binding to troponin C (TnC) and myosin binding to actin in activating thin filaments of rabbit psoas fibres. The ability of Ca 2+ to activate thin filaments was reduced by replacing native TnC with cardiac TnC (cTnC) or a site I-inactive skeletal TnC mutant (xsTnC). Acto-myosin (crossbridge) interaction was either inhibited using N -benzyl-p-toluene sulphonamide (BTS) or enhanced by lowering [ATP] from 5.0 to 0.5 mM. Reconstitution with cTnC reduced maximal force (F max ) by ∼1/3 and the Ca 2+ sensitivity of force (pCa 50 ) by 0.17 unit (P < 0.001), while reconstitution with xsTnC reduced F max by ∼2/3 and pCa 50 by 0.19 unit (P < 0.001). In both cases the apparent cooperativity of activation (n H ) was greatly decreased. In control fibres 3 µM BTS inhibited force to 57% of F max while in fibres reconstituted with cTnC or xsTnC, reconstituted maximal force (rF max ) was inhibited to 8.8% and 14.3%, respectively. Under control conditions 3 µM BTS significantly decreased the pCa 50 , but this effect was considerably reduced in cTnC reconstituted fibres, and eliminated in xsTnC reconstituted fibres. In contrast, when crossbridge cycle kinetics were slowed by lowering [ATP] from 5 to 0.5 mM in xsTnC reconstituted fibres, pCa 50 and n H were increased towards control values. Combined, our results demonstrate that when the ability of Ca 2+ binding to activate thin filaments is compromised, the relative contribution of strong crossbridges to maintain thin filament activation is increased. Furthermore, the data suggest that at low levels of Ca 2+ , the level of thin filament activation is determined primarily by the direct effects of Ca 2+ on tropomyosin mobility, while at higher levels of Ca 2+ the final level of thin filament activation is primarily determined by strong cycling crossbridges.

Cell transplantation improves cardiac function after myocardial infarction; however, the underlying mechanisms are not well-understood. Therefore, the goals of this study were to determine if neonatal rat cardiomyocytes transplanted into adult rat hearts one-week after infarction would, after 8-10 weeks: 1) improve global myocardial function, 2) contract in a Ca 2+ dependent manner, 3) influence mechanical properties of remote uninjured myocardium and 4) alter passive mechanical properties of infarct regions. The cardiomyocytes formed small grafts of ultrastructurally maturing myocardium that enhanced fractional shortening compared to non-treated infarcted hearts. Chemically demembranated tissue strips of cardiomyocyte grafts produced force when activated by Ca 2+ , whereas scar tissue did not. Furthermore, the Ca 2+ sensitivity of force was greater in cardiomyocyte grafts compared to control myocardium. Surprisingly, cardiomyocytes grafts isolated in the infarct zone increased Ca 2+ sensitivity of remote uninjured myocardium to levels greater than either remote myocardium from non-treated infarcted hearts or sham-operated controls. Enhanced calcium sensitivity was associated with decreased phosphorylation of cTnT, tropomyosin and MLC2, but not changes in myosin or troponin isoforms. Passive compliance of grafts resembled normal myocardium, while infarct tissue distant from grafts had compliance typical of scar. Thus, cardiomyocyte grafts are contractile, improve local tissue compliance and enhance calcium sensitivity of remote myocardium. Because the volume of remote myocardium greatly exceeds that of the grafts, this enhanced calcium sensitivity may be a major contributor to global improvements in ventricular function after cell transplantation.

The vision of a precision medicine–guided approach to novel cancer drug development is challenged by high intratumor heterogeneity and interpatient diversity. This complexity is rarely modeled accurately during preclinical drug development, hampering predictions of clinical drug efficacy. To address this issue, we developed Comparative In Vivo Oncology (CIVO) arrayed microinjection technology to test tumor responsiveness to simultaneous microdoses of multiple drugs directly in a patient’s tumor. Here, in a study of 18 canine patients with soft tissue sarcoma (STS), CIVO captured complex, patient-specific tumor responses encompassing both cancer cells and multiple immune infiltrates following localized exposure to different chemotherapy agents. CIVO also classified patient-specific tumor resistance to the most effective agent, doxorubicin, and further enabled assessment of a preclinical autophagy inhibitor, PS-1001, to reverse doxorubicin resistance. In a CIVO-identified subset of doxorubicin-resistant tumors, PS-1001 resulted in enhanced antitumor activity, increased infiltration of macrophages, and skewed this infiltrate toward M1 polarization. The ability to evaluate and cross-compare multiple drugs and drug combinations simultaneously in living tumors and across a diverse immunocompetent patient population may provide a foundation from which to make informed drug development decisions. This method also represents a viable functional approach to complement current precision oncology strategies.