在浸润性乳腺癌中 CAR 的表达及其对腺病毒转导效率的影响。

CAR expression in invasive breast carcinoma and its effect on adenovirus transduction efficiency.

机构信息

Moores Cancer Center, University of California San Diego, La Jolla, CA, 92037, USA.

Department of Chemistry and Biochemistry, University of California San Diego, La Jolla, CA, 92037, USA.

出版信息

Breast Cancer Res. 2024 Sep 10;26(1):131. doi: 10.1186/s13058-024-01880-z.

DOI:10.1186/s13058-024-01880-z

PMID:39256827

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11389499/

Abstract

BACKGROUND

Breast cancer is the second leading cause of death in women, with invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) as the two most common forms of invasive breast cancer. While estrogen receptor positive (ER+) IDC and ILC are treated similarly, the multifocality of ILC presents challenges in detection and treatment, worsening long-term clinical outcomes in patients. With increasing documentation of chemoresistance in ILC, additional treatment options are needed. Oncolytic adenoviral therapy may be a promising option, but cancer cells must express the coxsackievirus & adenovirus receptor (CAR) for adenoviral therapy to be effective. The present study aims to evaluate the extent to which CAR expression is observed in ILC in comparison to IDC, and how the levels of CAR expression correlate with adenovirus transduction efficiency. The effect of liposome encapsulation on transduction efficiency is also assessed.

METHODS

To characterize CAR expression in invasive breast carcinoma, 36 formalin-fixed paraffin-embedded (FFPE) human breast tumor samples were assayed by CAR immunohistochemistry (IHC). Localization of CAR in comparison to other junctional proteins was performed using a multiplex immunofluorescence panel consisting of CAR, p120-catenin, and E-cadherin. ILC and IDC primary tumors and cell lines were transduced with E1- and E3-deleted adenovirus type 5 inserted with a GFP transgene (Ad-GFP) and DOTAP liposome encapsulated Ad-GFP (DfAd-GFP) at various multiplicities of infection (MOIs). Transduction efficiency was measured using a fluorescence plate reader. CAR expression in the human primary breast carcinomas and cell lines was also evaluated by IHC.

RESULTS

We observed membranous CAR, p120-catenin and E-cadherin expression in IDC. In ILC, we observed cytoplasmic expression of CAR and p120-catenin, with absent E-cadherin. Adenovirus effectively transduced high-CAR IDC cell lines, at MOIs as low as 12.5. Ad-GFP showed similar transduction as DfAd-GFP in high-CAR IDC cell lines. Conversely, Ad-GFP transduction of ILC cell lines was observed only at MOIs of 50 and 100. Furthermore, Ad-GFP did not transduce CAR-negative IDC cell lines even at MOIs greater than 100. Liposome encapsulation (DfAd-GFP) improved transduction efficiency 4-fold in ILC and 17-fold in CAR-negative IDC cell lines.

CONCLUSION

The present study demonstrates that oncolytic adenoviral therapy is less effective in ILC than IDC due to differences in spatial CAR expression. Liposome-enhanced delivery may be beneficial for patients with ILC and tumors with low or negative CAR expression to improve adenoviral therapeutic effectiveness.

摘要

背景

乳腺癌是女性死亡的第二大主要原因，其中浸润性导管癌（IDC）和浸润性小叶癌（ILC）是两种最常见的浸润性乳腺癌。虽然雌激素受体阳性（ER+）IDC 和 ILC 的治疗方式相似，但 ILC 的多灶性在检测和治疗方面带来了挑战，导致患者的长期临床结局恶化。随着越来越多的证据表明 ILC 存在化疗耐药性，需要更多的治疗选择。溶瘤腺病毒治疗可能是一种有前途的选择，但癌细胞必须表达柯萨奇病毒和腺病毒受体（CAR），溶瘤腺病毒治疗才会有效。本研究旨在评估 CAR 在 ILC 中的表达程度与 IDC 的比较，并研究 CAR 表达水平与腺病毒转导效率的相关性。还评估了脂质体包封对转导效率的影响。

方法

为了研究 CAR 在浸润性乳腺癌中的表达情况，我们通过 CAR 免疫组化（IHC）检测了 36 例福尔马林固定石蜡包埋（FFPE）的人乳腺肿瘤样本。通过使用包含 CAR、p120-连环蛋白和 E-钙黏蛋白的多重免疫荧光试剂盒，比较了 CAR 在细胞中的定位与其他连接蛋白的定位。我们使用 E1 和 E3 缺失的腺病毒 5 型（插入 GFP 转基因）（Ad-GFP）和 DOTAP 脂质体包封的 Ad-GFP（DfAd-GFP），在不同的感染复数（MOI）下转导 ILC 和 IDC 原发性肿瘤和细胞系。使用荧光板读数器测量转导效率。还通过 IHC 评估了人原发性乳腺癌和细胞系中的 CAR 表达情况。

结果

我们观察到 IDC 中有膜结合的 CAR、p120-连环蛋白和 E-钙黏蛋白的表达。在 ILC 中，我们观察到 CAR 和 p120-连环蛋白的细胞质表达，而 E-钙黏蛋白缺失。腺病毒在 MOI 低至 12.5 时，有效转导高 CAR IDC 细胞系。高 CAR IDC 细胞系中，Ad-GFP 和 DfAd-GFP 的转导效果相似。相反，Ad-GFP 仅在 MOI 为 50 和 100 时转导 ILC 细胞系。此外，即使 MOI 大于 100，腺病毒也不能转导 CAR 阴性的 IDC 细胞系。脂质体包封（DfAd-GFP）使 ILC 和 CAR 阴性 IDC 细胞系的转导效率分别提高了 4 倍和 17 倍。