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Microdissected "cuboids" for microfluidic drug testing of intact tissues

Lab Chip

2020 Gargi Mishra; Albert Folch; Taranjit Gujral; Robert Pierce

As preclinical animal tests often do not accurately predict drug effects later observed in humans, most drugs under development fail to reach the market. Thus there is a critical need for functional drug testing platforms that use human, intact tissues to complement animal studies. To enable future multiplexed delivery of many drugs to one small biopsy, we have developed a multi-well microfluidic platform that selectively treats cuboidal-shaped microdissected tissues or "cuboids" with well-preserved tissue microenvironments. We create large numbers of uniformly-sized cuboids by semi-automated sectioning of tissue with a commercially available tissue chopper. Here we demonstrate the microdissection method on normal mouse liver, which we characterize with quantitative 3D imaging, and on human glioma xenograft tumors, which we evaluate after time in culture for viability and preservation of the microenvironment. The benefits of size uniformity include lower heterogeneity in future biological assays as well as facilitation of their physical manipulation by automation. Our prototype platform consists of a microfluidic circuit whose hydrodynamic traps immobilize the live cuboids in arrays at the bottom of a multi-well plate. Fluid dynamics simulations enabled the rapid evaluation of design alternatives and operational parameters. We demonstrate the proof-of-concept application of model soluble compounds such as dyes (CellTracker, Hoechst) and the cancer drug cisplatin. Upscaling of the microfluidic platform and microdissection method to larger arrays and numbers of cuboids could lead to direct testing of human tissues at high throughput, and thus could have a significant impact on drug discovery and personalized medicine.

Drug-drug Interactions in Patients with HIV and Cancer in Sub-Saharan Africa


2020 Thomas Uldrick

In Sub-Saharan Africa, the cancer burden is predicted to increase by > 85% by 2030, the largest increase worldwide. This region has a large HIV-positive population. Drug-drug interactions (DDIs) from concomitant use of multiple drugs increase the risk of drug toxicities, sub-optimal therapy, and drug resistance. With the increase in polypharmacy, involving antiretroviral (ARV), and anticancer drugs, there is a greater need for an appreciation of clinically relevant DDIs. Anticancer and ARV drugs studied in this review were from The World Health Organization's Model List of Essential Medicines 2017. We reviewed; drug package inserts, <a href=""></a>&nbsp;and <a href=""></a>, to evaluate pharmacokinetic interactions with cytochrome P450 (CYP450) and ABCB1. The DDIs between drugs were assessed using the University Of Liverpool, UK HIV Drug Interactions Checker, and the LexiComp Drug Interaction tool of <a href=""></a>. About 70% of ARVs studied interact with CYP450, all involve CYP3A4, and 55% interact with ABCB1. About 65% of anticancer drugs interact with CYP450, 44% of which do so through CYP3A4. About 75% of anticancer drugs interact with ARV drugs, with nine absolute contraindications to concomitant therapy. There exist a substantial number of DDIs between ARV and anticancer drugs, primarily mediated through CYP450 enzymes. Dolutegravir based regimens offer the safest DDI profile for concurrent use with anticancer drugs. However, there are substantial gaps in our knowledge, and this study serves to highlight the need for additional research to better define these interactions and their effect on drug exposure, as attention to these DDIs is a relatively simple intervention that could lead to optimizing disease treatment.

Genetically engineered macrophages persist in solid tumors and locally deliver therapeutic proteins to activate immune responses

J Immunother Cancer

2020 Chibawanye Ene; Michael Jensen; Kimberly Smythe; Jean Campbell; Courtney Crane; Eric Holland; Venu Pillarisetty; Robert Pierce

BACKGROUND: Though currently approved immunotherapies, including chimeric antigen receptor T cells and checkpoint blockade antibodies, have been successfully used to treat hematological and some solid tumor cancers, many solid tumors remain resistant to these modes of treatment. In solid tumors, the development of effective antitumor immune responses is hampered by restricted immune cell infiltration and an immunosuppressive tumor microenvironment (TME). An immunotherapy that infiltrates and persists in the solid TME, while providing local, stable levels of therapeutic to activate or reinvigorate antitumor immunity could overcome these challenges faced by current immunotherapies. METHODS: Using lentivirus-driven engineering, we programmed human and murine macrophages to express therapeutic payloads, including Interleukin (IL)-12. In vitro coculture studies were used to evaluate the effect of genetically engineered macrophages (GEMs) secreting IL-12 on T cells and on the GEMs themselves. The effects of IL-12 GEMs on gene expression profiles within the TME and tumor burden were evaluated in syngeneic mouse models of glioblastoma and melanoma and in human tumor slices isolated from patients with advanced gastrointestinal malignancies. RESULTS: Here, we present a cellular immunotherapy platform using lentivirus-driven genetic engineering of human and mouse macrophages to constitutively express proteins, including secreted cytokines and full-length checkpoint antibodies, as well as cytoplasmic and surface proteins that overcomes these barriers. GEMs traffic to, persist in, and express lentiviral payloads in xenograft mouse models of glioblastoma, and express a non-signaling truncated CD19 surface protein for elimination. IL-12-secreting GEMs activated T cells and induced interferon-gamma (IFN) in vitro and slowed tumor growth resulting in extended survival in vivo. In a syngeneic glioblastoma model, IFN signaling cascades were also observed in mice treated with mouse bone-marrow-derived GEMs secreting murine IL-12. These findings were reproduced in ex vivo tumor slices comprised of intact MEs. In this setting, IL-12 GEMs induced tumor cell death, chemokines and IFN-stimulated genes and proteins. CONCLUSIONS: Our data demonstrate that GEMs can precisely deliver titratable doses of therapeutic proteins to the TME to improve safety, tissue penetrance, targeted delivery and pharmacokinetics.

Anti-PD-1 and Anti-PD-L1 Monoclonal Antibodies in People Living with HIV and Cancer


2020 Thomas Uldrick

PURPOSE OF REVIEW: Immune checkpoint inhibitors targeting the programmed cell death-1 (PD-1) pathway are a class of anti-cancer immunotherapy agents changing treatment paradigms of many cancers that occur at higher rates in people living with HIV (PLWH) than in the general population. However, PLWH have been excluded from most of the initial clinical trials with these agents. RECENT FINDINGS: Two recent prospective studies of anti-PD-1 agents, along with observational studies and a meta-analysis, have demonstrated acceptable safety in PLWH. Preliminary evidence indicates activity in a range of tumors and across CD4+ T cell counts. Safety and preliminary activity data suggest monoclonal antibodies targeting PD-1 or its ligand, PD-L1, are generally appropriate for PLWH and cancers for which there are FDA-approved indications. Ongoing and future trials of anti-PD-1 and anti-PD-L1 therapy alone or in combination for HIV-associated cancers may further improve outcomes for this underserved population.

CAR T-cell therapy for cancer and HIV through novel approaches to HIV-associated haematological malignancies

Lancet Haematol

2020 Hans-Peter Kiem; Thomas Uldrick; Blake Rust

People living with HIV are a global population with increased cancer risk but their access to modern immunotherapies for cancer treatment has been limited by socioeconomic factors and inadequate research to support safety and efficacy in this population. These immunotherapies include immune checkpoint inhibitors and advances in cellular immunotherapy, particularly chimeric antigen receptor (CAR) T-cell therapy. Despite the field of cancer immunotherapy rapidly expanding with ongoing clinical trials, people with HIV are often excluded from such trials. In 2019, post-approval evaluation of anti-CD19 CAR T-cell therapy in people with HIV and aggressive B-cell lymphoma showed the feasibility of CAR T-cell therapy for cancer in this excluded group. Along with expanded treatment options for people with HIV is the ability to assess the effects of immunotherapy on the latent HIV reservoir, with certain immunotherapies showing the ability to alleviate this burden. This Series paper addresses the increased cancer burden in people with HIV, the increasing evidence for the safety and efficacy of immunotherapies in the context of HIV and cancer, and opportunities for novel applications of CAR-T therapy for the treatment of both haematological malignancies and HIV.

Treatment of HIV-associated Primary CNS Lymphoma with Antiretroviral Therapy, Rituximab, and High-Dose Methotrexate


2020 Thomas Uldrick


Tocilizumab in patients with symptomatic Kaposi sarcoma herpesvirus-associated multicentric Castleman disease


2020 Thomas Uldrick


Signatures of oral microbiome in HIV-infected individuals with oral Kaposi's sarcoma and cell-associated KSHV DNA

PLoS Pathog

2020 Thomas Uldrick

Infection by Kaposi's sarcoma-associated herpesvirus (KSHV) is necessary for the development of Kaposi's sarcoma (KS), which most often develops in HIV-infected individuals. KS frequently has oral manifestations and KSHV DNA can be detected in oral cells. Numerous types of cancer are associated with the alteration of microbiome including bacteria and virus. We hypothesize that oral bacterial microbiota affects or is affected by oral KS and the presence of oral cell-associated KSHV DNA. In this study, oral and blood specimens were collected from a cohort of HIV/KSHV-coinfected individuals all previously diagnosed with KS, and were classified as having oral KS with any oral cell-associated KSHV DNA status (O-KS, n = 9), no oral KS but with oral cell-associated KSHV DNA (O-KSHV, n = 10), or with neither oral KS nor oral cell-associated KSHV DNA (No KSHV, n = 10). We sequenced the hypervariable V1-V2 region of the 16S rRNA gene present in oral cell-associated DNA by next generation sequencing. The diversity, richness, relative abundance of operational taxonomic units (OTUs) and taxonomic composition of oral microbiota were analyzed and compared across the 3 studied groups. We found impoverishment of oral microbial diversity and enrichment of specific microbiota in O-KS individuals compared to O-KSHV or No KSHV individuals. These results suggest that HIV/KSHV coinfection and oral microbiota might impact one another and influence the development of oral KS.

Intratumoral Plasmid IL12 Electroporation Therapy in Patients with Advanced Melanoma Induces Systemic and Intratumoral T-cell Responses

Cancer Immunol Res

2020 Robert Pierce

Whereas systemic IL-12 is associated with potentially life-threatening toxicity, intra-tumoral delivery of IL-12 through tavokinogene telseplasmid electroporation (tavo) is safe and can induce tumor regression at distant sites. The mechanism by which these responses are mediated is unknown, but is presumed to result from a cellular immune response. In a phase II clinical trial of tavo (NCT01502293), samples from 28 cutaneous melanoma patients with in-transit disease were assessed for immune responses induced with this treatment. Within the blood circulating immune cell population, we found that the frequencies of circulating PD-1+ CD4+ and CD8+ T cells declined with treatment. Circulating immune responses to gp100 were also detected following treatment as measured by IFN-γ ELISPOT. Patients with a greater antigen-specific circulating immune response also had higher numbers of CD8+ T cells within the tumor. Clinical response was also associated with increased intratumoral CD3+ T cells. Finally, intratumoral T cell clonality and convergence were increased after treatment, indicating a focusing of the TCR repertoire. These results indicated that local treatment with tavo can induce a systemic T cell response and recruit T cells to the tumor microenvironment.

Identifying and targeting pathogenic PI3K/AKT/mTOR signaling in IL-6 blockade-refractory idiopathic multicentric Castleman disease

J Clin Invest

2019 Thomas Uldrick

BACKGROUND: Idiopathic multicentric Castleman disease (iMCD) is a hematologic illness involving cytokine-induced lymphoproliferation, systemic inflammation, cytopenias, and life-threatening multi-organ dysfunction. The molecular underpinnings of interleukin-6(IL-6)-blockade refractory patients remain unknown; no targeted therapies exist. In this study, we searched for therapeutic targets in IL-6-blockade refractory iMCD patients with the thrombocytopenia, anasarca, fever/elevated C-reactive protein, reticulin myelofibrosis, renal dysfunction, organomegaly (TAFRO) clinical subtype. METHODS: We analyzed tissues and blood samples from three IL-6-blockade refractory iMCD-TAFRO patients. Cytokine panels, quantitative serum proteomics, flow cytometry of PBMCs, and pathway analyses were employed to identify novel therapeutic targets. To confirm elevated mTOR signaling, a candidate therapeutic target from the above assays, immunohistochemistry was performed for phosphorylated S6, a read-out of mTOR activation, in three iMCD lymph node tissue samples and controls. Proteomic, immunophenotypic, and clinical response assessments were performed to quantify the effects of administration of the mTOR inhibitor, sirolimus. RESULTS: Studies of three IL-6-blockade refractory iMCD cases revealed increased CD8+ T cell activation, VEGF-A, and PI3K/Akt/mTOR pathway activity. Administration of sirolimus significantly attenuated CD8+ T cell activation and decreased VEGF-A levels. Sirolimus induced clinical benefit responses in all three patients with durable and ongoing remissions of 66, 19, and 19 months. CONCLUSION: This precision medicine approach identifies PI3K/Akt/mTOR signaling as the first pharmacologically-targetable pathogenic process in IL-6-blockade refractory iMCD. Prospective evaluation of sirolimus in treatment-refractory iMCD is planned (NCT03933904). FUNDING: Castleman's Awareness & Research Effort/Castleman Disease Collaborative Network, Penn Center for Precision Medicine, University Research Foundation, Intramural NIH funding, and National Heart Lung and Blood Institute.