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Heightened sensitivity of forensic DNA techniques has led to an increased variety of samples tested, often yielding complex DNA mixtures, in turn making the interpretation of profiling results more complicated. Currently, there is no prescribed upstream laboratory method to separate complex DNA mixtures by their contributors; therefore, a method is needed that could reduce mixtures into their component parts. Various cell sorting applications have the potential to be this method, if intact cells can be reliably obtained from forensic samples. Here, the effects of elution buffer and swab substrate on the recovery of intact, human, white blood cells from dried blood samples were evaluated. Approximately 328,000 cells per swab were deposited onto cotton, flocked, and dissolvable swabs. The whole-cell elution of the dried samples was evaluated with water, phosphate buffered saline, and AutoMACS® elution buffers. We demonstrate that AutoMACS® buffer is superior for the elution of intact cells, compared to phosphate buffered saline and water. When swab type was considered, the highest yield of intact cells resulted from flocked swabs, as opposed to cotton or dissolvable swabs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The HER family of receptor tyrosine kinases has been linked to deregulation of growth and proliferation for multiple types of cancer. Members have therefore become thefocus of many drug and immune-based therapy innovations. The targeted anti-cancer agent, lapatinib, is a small molecule inhibitor that directly interferes with EGFR (HER-1)and HER-2 signaling, and indirectly reduces HER-3 signaling, thus suppressing important downstream events. A recently-developed dendritic cell-based vaccine against early breast cancer (ductal carcinoma in situ; DCIS) that generates strong Th1-dominated immunity against HER-2 has induced pathologic complete response in about one-third of immunized individuals. In vitro studies suggested cytokines secreted by Th1 cells could be major contributors to the vaccine effects including induction of apoptosis and suppression of HER expression. With a view toward improving complete response rates, we investigated whether the principle Th1 cytokines (IFN-γ and TNF-α) could act in concert with lapatinib to suppress activity of breast cancer lines in vitro. Lapatinib-sensitive SKBR3, MDA-MB-468 and BT474 cells were incubated with Th1 cytokines, lapatinib, or both. It was found that combined treatment maximized metabolic suppression(Alamar Blue assay), as well as cell death (Trypan Blue) and apoptosis(Annexin V/Propidium Iodide and TMRE staining). Combined drug plus cytokine treatment also maximized suppression of both total and phosphorylated forms of HER-2 and HER-3. Interestingly, when lapatinib resistant lines MDA-MB-453 and JIMT-1 were tested, it was found that the presence of Th1 cytokines appeared to enhance sensitivity for lapatinib-induced metabolic suppression and induction of apoptotic cell death, nearly abrogating drug resistance. These studies provide pre-clinical data suggesting the possibility that targeted drug therapy may be combined with vaccination to enhance anti-cancer effects, and furthermore that robust immunity in the form of secreted Th1 cytokines may have the capacity to mitigate resistance to targeted drugs., Competing Interests: The authors of this manuscript have the following competing interests: 1) Patent filings (BJC, GKK, LS; USPTO # 62/741,664: Combination Therapy For Treating Cancer filed 10/5/18) and 2); Membership on an advisory board for Immunorestoration LLC (BJC and GKK). This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

HER2-directed therapies are less effective in patients with ER pos compared to ER neg breast cancer, possibly reflecting bidirectional activation between HER2 and estrogen signaling pathways. We investigated dual blockade using anti-HER2 vaccination and anti-estrogen therapy in HER2 pos /ER pos early breast cancer patients. In pre-clinical studies of HER2 pos breast cancer cell lines, ER pos cells were partially resistant to CD4 + Th1 cytokine-induced metabolic suppression compared with ER neg cells. The addition of anti-estrogen treatment significantly enhanced cytokine sensitivity in ER pos , but not ER neg , cell lines. In two pooled phase-I clinical trials, patients with HER2 pos early breast cancer were treated with neoadjuvant anti-HER2 dendritic cell vaccination; HER2 pos /ER pos patients were treated with or without concurrent anti-estrogen therapy. The anti-HER2 Th1 immune response measured in the peripheral blood significantly increased following vaccination, but was similar across the three treatment groups (ER neg vaccination alone, ER pos vaccination alone, ER pos vaccination + anti-estrogen therapy). In the sentinel lymph nodes, however, the anti-HER2 Th1 immune response was significantly higher in ER pos patients treated with combination anti-HER2 vaccination plus anti-estrogen therapy compared to those treated with anti-HER2 vaccination alone. Similar rates of pathologic complete response (pCR) were observed in vaccinated ER neg patients and vaccinated ER pos patients treated with concurrent anti-estrogen therapy (31.4% vs. 28.6%); both were significantly higher than the pCR rate in vaccinated ER pos patients who did not receive anti-estrogen therapy (4.0%, p = 0.03). Since pCR portends long-term favorable outcomes, these results support additional clinical investigations using HER2-directed vaccines in combination with anti-estrogen treatments for ER pos /HER2 pos DCIS and invasive breast cancer.